What's hot today:
Current papers in developmental biology and gene function


Tuesday June 30, 2020 - Behavior

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De Jesus-Olmo, L. A., Rodriguez, N., Francia, M., Aleman-Rios, J., Pacheco-Agosto, C. J., Ortega-Torres, J., Nieves, R., Fuenzalida-Uribe, N., Ghezzi, A. and Agosto, J. L. (2020). Pumilio Regulates Sleep Homeostasis in Response to Chronic Sleep Deprivation in Drosophila melanogaster. Front Neurosci 14: 319. PubMed ID: 32362810
Recent studies have identified the Drosophila brain circuits involved in the sleep/wake switch and have pointed to the modulation of neuronal excitability as one of the underlying mechanisms triggering sleep need. This study aimed to explore the link between the homeostatic regulation of neuronal excitability and sleep behavior in the circadian circuit. For this purpose, this study chose Pumilio (Pum), whose main function is to repress protein translation and has been linked to modulation of neuronal excitability during chronic patterns of altered neuronal activity. This study explored the effects of Pum on sleep homeostasis in Drosophila melanogaster, which shares most of the major features of mammalian sleep homeostasis. The evidence indicates that Pum is necessary for sleep rebound and that its effect is more pronounced during chronic sleep deprivation (84 h) than acute deprivation (12 h). Knockdown of pum, results in a reduction of sleep rebound during acute sleep deprivation and the complete abolishment of sleep rebound during chronic sleep deprivation. Based on these findings, it is proposed that Pum is a critical regulator of sleep homeostasis through neural adaptations triggered during sleep deprivation.
Jezovit, J. A., Rooke, R., Schneider, J. and Levine, J. D. (2020). Behavioral and environmental contributions to drosophilid social networks. Proc Natl Acad Sci U S A 117(21): 11573-11583. PubMed ID: 32404421
Animals interact with each other in species-specific reproducible patterns. These patterns of organization are captured by social network analysis, and social interaction networks (SINs) have been described for a wide variety of species including fish, insects, birds, and mammals. The aim of this study is to understand the evolution of social organization in Drosophila. Using a comparative ecological, phylogenetic, and behavioral approach, the different properties of SINs formed by 20 drosophilids were compared. Whether drosophilid network structures arise from common ancestry, a response to the species' past climate, other social behaviors, or a combination of these factors was investigated. This study shows that differences in past climate predicted the species' current SIN properties. The drosophilid phylogeny offered no value to predicting species' differences in SINs through phylogenetic signal tests. This suggests that group-level social behaviors in drosophilid species are shaped by divergent climates. However, it was found that the social distance at which flies interact correlated with the drosophilid phylogeny, indicating that behavioral elements of SINs have remained largely unchanged in their evolutionary history. A significant correlation was found of leg length to social distance, outlining the interdependence of anatomy and complex social structures. Although SINs display a complex evolutionary relationship across drosophilids, this study suggests that the ecology, and not common ancestry, contributes to diversity in social structure in Drosophila.
Wiggin, T. D., Goodwin, P. R., Donelson, N. C., Liu, C., Trinh, K., Sanyal, S. and Griffith, L. C. (2020). Covert sleep-related biological processes are revealed by probabilistic analysis in Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 32303656
Sleep pressure and sleep depth are key regulators of wake and sleep. Current methods of measuring these parameters in Drosophila melanogaster have low temporal resolution and/or require disrupting sleep. This study reports analysis tools for high-resolution, noninvasive measurement of sleep pressure and depth from movement data. Probability of initiating activity, P(Wake), measures sleep depth while probability of ceasing activity, P(Doze), measures sleep pressure. In vivo and computational analyses show that P(Wake) and P(Doze) are largely independent and control the amount of total sleep. A Hidden Markov Model was developed that allows visualization of distinct sleep/wake substates. These hidden states have a predictable relationship with P(Doze) and P(Wake), suggesting that the methods capture the same behaviors. Importantly, this study demonstrates that both the Doze/Wake probabilities and the sleep/wake substates are tied to specific biological processes. These metrics provide greater mechanistic insight into behavior than measuring the amount of sleep alone.
Wohl, M., Ishii, K. and Asahina, K. (2020). Layered roles of fruitless isoforms in specification and function of male aggression-promoting neurons in Drosophila. Elife 9. PubMed ID: 32314957
Inter-male aggressive behavior is a prominent sexually dimorphic behavior. Neural circuits that underlie aggressive behavior are therefore likely under the control of sex-determining genes. However, the neurogenetic mechanism that generates sex-specific aggressive behavior remains largely unknown. This study found that a neuronal class specified by one of the Drosophila sex determining genes, fruitless (fru), belongs to the neural circuit that generates male-type aggressive behavior. This neuronal class can promote aggressive behavior independent of another sex determining gene, doublesex (dsx), although dsx is involved in ensuring that aggressive behavior is performed only toward males. This study also found that three fru isoforms with different DNA binding domains show a division of labor on male aggressive behaviors. A dominant role of fru in specifying sex-specific aggressive behavior may underscore a genetic mechanism that allows male-type aggressive behavior to evolve at least partially independently from courtship behavior, which is under different selective pressures.
Wu, F., Deng, B., Xiao, N., Wang, T., Li, Y., Wang, R., Shi, K., Luo, D. G., Rao, Y. and Zhou, C. (2020). A neuropeptide regulates fighting behavior in Drosophila melanogaster. Elife 9. PubMed ID: 32314736
Aggressive behavior is regulated by various neuromodulators such as neuropeptides and biogenic amines. This study found that the neuropeptide Drosulfakinin (Dsk) modulates aggression in Drosophila melanogaster. Knock-out of Dsk or Dsk receptor CCKLR-17D1 reduced aggression. Activation and inactivation of Dsk-expressing neurons increased and decreased male aggressive behavior, respectively. Moreover, data from transsynaptic tracing, electrophysiology and behavioral epistasis reveal that Dsk-expressing neurons function downstream of a subset of P1 neurons (P1(a)-splitGAL4) to control fighting behavior. In addition, winners show increased calcium activity in Dsk-expressing neurons. Conditional overexpression of Dsk promotes social dominance, suggesting a positive correlation between Dsk signaling and winning effects. The mammalian ortholog CCK has been implicated in mammal aggression, thus this work suggests a conserved neuromodulatory system for the modulation of aggressive behavior.
Charroux, B., Daian, F. and Royet, J. (2020). Drosophila Aversive Behavior toward Erwinia carotovora carotovora Is Mediated by Bitter Neurons and Leukokinin. iScience 23(6): 101152. PubMed ID: 32450516
The phytopathogen Erwinia carotovora carotovora (Ecc) has been used successfully to decipher some of the mechanisms that regulate the interactions between Drosophila melanogaster and bacteria, mostly following forced association between the two species. How do Drosophila normally perceive and respond to the presence of Ecc is unknown. Using a fly feeding two-choice assay and video tracking, this study shows that Drosophila are first attracted but then repulsed by an Ecc-contaminated solution. The initial attractive phase is dependent on the olfactory Gr63a and Gαq proteins, whereas the second repulsive phase requires a functional gustatory system. Genetic manipulations and calcium imaging indicate that bitter neurons and gustatory receptors Gr66a and Gr33a are needed for the aversive phase and that the neuropeptide leukokinin is also involved. This study also demonstrates that these behaviors are independent of the NF-κB cascade that controls some of the immune, metabolic, and behavioral responses to bacteria.

Monday, June 29th - Chromatin

Meyer-Nava, S., Torres, A., Zurita, M. and Valadez-Graham, V. (2020). Molecular effects of dADD1 misexpression in chromatin organization and transcription. BMC Mol Cell Biol 21(1): 17. PubMed ID: 32293240
dADD1 and dXNP proteins are the orthologs in Drosophila melanogaster of the ADD and SNF2 domains, respectively, of the ATRX vertebrate's chromatin remodeler, they suppress position effect variegation phenotypes and participate in heterochromatin maintenance. This study performed a search in human cancer databases and found that ATRX protein levels were elevated in more than 4.4% of the samples analyzed. Using the Drosophila model, the effects of over and under-expression of dADD1 proteins was tested in polytene cells. Elevated levels of dADD1 in fly tissues caused different phenotypes, such as chromocenter disruption and loss of banding pattern at the chromosome arms. Analyses of the heterochromatin maintenance protein HP1a, the dXNP ATPase and the histone post-translational modification H3K9me3 revealed changes in their chromatin localization accompanied by mild transcriptional defects of genes embedded in heterochromatic regions. Furthermore, the expression of heterochromatin embedded genes in null dadd1 organisms is lower than in the wild-type conditions. These data indicate that dADD1 overexpression induces chromatin changes, probably affecting the stoichiometry of HP1a containing complexes that lead to transcriptional and architectural changes. These results place dADD1 proteins as important players in the maintenance of chromatin architecture and heterochromatic gene expression.
Ilyin, A. A., Stolyarenko, A. D., Klenov, M. S. and Shevelyov, Y. Y. (2020). Various modes of HP1a interactions with the euchromatic chromosome arms in Drosophila ovarian somatic cells. Chromosoma. PubMed ID: 32500264
Heterochromatin protein 1a (HP1a) is a well-known component of pericentromeric and telomeric heterochromatin in Drosophila. However, its role and the mechanisms of its binding in the chromosome arms (ChAs) remain largely unclear. This study identified HP1a-interacting domains in the somatic cells of Drosophila ovaries using a DamID-seq approach and compared them with insertion sites of transposable elements (TEs) revealed by genome sequencing. Although HP1a domains cover only 13% of ChAs, they non-randomly associate with 42% of TE insertions. Furthermore, HP1a on average propagates at 2-kb distances from the TE insertions. These data confirm the role of TEs in formation of HP1a islands in ChAs. However, only 18% of HP1a domains have adjacent TEs, indicating the existence of other mechanisms of HP1a domain formation besides spreading from TEs. In particular, many TE-independent HP1a domains correspond to the regions attached to the nuclear pore complexes (NPCs) or contain active gene promoters. However, HP1a occupancy on the promoters does not significantly influence expression of corresponding genes. At the same time, the steady-state transcript level of many genes located outside of HP1a domains was altered upon HP1a knockdown in the somatic cells of ovaries, thus pointing to the strong indirect effect of HP1a depletion. Collectively, these results support an existence of at least three different mechanisms of HP1a domain emergence in ChAs: spreading from TE insertions, transient interactions with the chromatin located near NPCs, and targeting to the promoters of moderately expressed genes.
Bobkov, G. O. M., Huang, A., van den Berg, S. J. W., Mitra, S., Anselm, E., Lazou, V., Schunter, S., Feederle, R., Imhof, A., Lusser, A., Jansen, L. E. T. and Heun, P. (2020). Spt6 is a maintenance factor for centromeric CENP-A. Nat Commun 11(1): 2919. PubMed ID: 32522980
Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This presents both an opportunity to remodel the underlying chromatin and a danger of losing epigenetic information. Centromeric transcription is required for stable incorporation of the centromere-specific histone dCENP-A in M/G1 phase, which depends on the eviction of previously deposited H3/H3.3-placeholder nucleosomes. This study demonstrates that the histone chaperone and transcription elongation factor Spt6 spatially and temporarily coincides with centromeric transcription and prevents the loss of old CENP-A nucleosomes in both Drosophila and human cells. Spt6 binds directly to dCENP-A and dCENP-A mutants carrying phosphomimetic residues alleviate this association. Retention of phosphomimetic dCENP-A mutants is reduced relative to wildtype, while non-phosphorylatable dCENP-A retention is increased and accumulates at the centromere. It is concluded that Spt6 acts as a conserved CENP-A maintenance factor that ensures long-term stability of epigenetic centromere identity during transcription-mediated chromatin remodeling.
Gaub, A., Sheikh, B. N., Basilicata, M. F., Vincent, M., Nizon, M., Colson, C., Bird, M. J., Bradner, J. E., Thevenon, J., Boutros, M. and Akhtar, A. (2020). Evolutionary conserved NSL complex/BRD4 axis controls transcription activation via histone acetylation. Nat Commun 11(1): 2243. PubMed ID: 32382029
Cells rely on a diverse repertoire of genes for maintaining homeostasis, but the transcriptional networks underlying their expression remain poorly understood. The MOF acetyltransferase-containing Non-Specific Lethal (NSL) complex is a broad transcription regulator. It is essential in Drosophila, and haploinsufficiency of the human KANSL1 subunit results in the Koolen-de Vries syndrome. This study, performed a genome-wide RNAi screen and identify the BET protein BRD4 (see Drosophila Female sterile (1) homeotic) as an evolutionary conserved co-factor of the NSL complex. Using Drosophila and mouse embryonic stem cells, this study characterised a recruitment hierarchy, where NSL-deposited histone acetylation enables BRD4 recruitment for transcription of constitutively active genes. Transcriptome analyses in Koolen-de Vries patient-derived fibroblasts reveals perturbations with a cellular homeostasis signature that are evoked by the NSL complex/BRD4 axis. It is proposed that BRD4 represents a conserved bridge between the NSL complex and transcription activation, and provide a new perspective in the understanding of their functions in healthy and diseased states.
Lee, Y. C. G., Ogiyama, Y., Martins, N. M. C., Beliveau, B. J., Acevedo, D., Wu, C. T., Cavalli, G. and Karpen, G. H. (2020). Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin. PLoS Genet 16(3): e1008673. PubMed ID: 32203508
Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. This study investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, this study reveals a hierarchical organization of the PCH domains into distinct "territories." Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. These findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin.
Dou, K., Liu, Y., Zhang, Y., Wang, C., Huang, Y. and Zhang, Z. Z. (2020). Drosophila P75 safeguards oogenesis by preventing H3K9me2 spreading. J Genet Genomics. PubMed ID: 32499180
Serving as a host factor for human immunodeficiency virus (HIV) integration, LEDGF/p75 has been under extensive study as a potential target for therapy. However, as a highly conserved protein, its physiological function remains to be thoroughly elucidated. This study characterized the molecular function of dP75, the Drosophila homolog of LEDGF/p75, during oogenesis. dP75 binds to transcriptionally active chromatin with its PWWP domain. The C-terminus integrase-binding domain-containing region of dP75 physically interacts with the histone kinase Jil-1 and stabilizes it in vivo. Together with Jil-1, dP75 prevents the spreading of the heterochromatin mark-H3K9me2-onto genes required for oogenesis and piRNA production. Without dP75, ectopical silencing of these genes disrupts oogenesis, activates transposons, and causes animal sterility. It is proposed that dP75, the homolog of an HIV host factor in Drosophila, partners with and stabilizes Jil-1 to ensure gene expression during oogenesis by preventing ectopic heterochromatin spreading.

Friday, June 26th - Disease Models

Hidalgo, S., Castro, C., Zarate, R. V., Valderrama, B. P., Hodge, J. J. L. and Campusano, J. M. (2020). The behavioral and neurochemical characterization of a Drosophila dysbindin mutant supports the contribution of serotonin to schizophrenia negative symptoms. Neurochem Int 138: 104753. PubMed ID: 32416114
Mutations in the dystrobrevin binding protein 1 (DTNBP1) gene that encodes for the dysbindin-1 protein, are associated with a higher risk for schizophrenia. Interestingly, individuals carrying high-risk alleles in this gene have been associated with an increased incidence of negative symptoms for the disease, which include anhedonia, avolition and social withdrawal. This study evaluated behavioral and neurochemical changes in a hypomorphic Drosophila mutant for the orthologue of human Dysbindin-1, dysb1. Mutant dysb1 flies exhibit altered social space parameters, suggesting asocial behavior, accompanied by reduced olfactory performance. Moreover, dysb1 mutant flies show poor performance in basal and startle-induced locomotor activity. This study also reports a reduction in serotonin brain levels and changes in the expression of the Drosophila serotonin transporter (dSERT) in dysb1 flies. The data show that the serotonin-releasing amphetamine derivative 4-methylthioamphetamine (4-MTA) modulates social spacing and locomotion in control flies, suggesting that serotonergic circuits modulate these behaviors. 4-MTA was unable to modify the behavioral deficiencies in mutant flies, which is consistent with the idea that the efficiency of pharmacological agents acting at dSERT depends on functional serotonergic circuits. Thus, these data show that the dysb1 mutant exhibits behavioral deficits that mirror some aspects of the endophenotypes associated with the negative symptoms of schizophrenia. It is argued that at least part of the behavioral aspects associated with these symptoms could be explained by a serotonergic deficit. The dysb1 mutant presents an opportunity to study the molecular underpinnings of schizophrenia negative symptoms and reveals new potential targets for treatment of the disease.
Elmen, L., Volpato, C. B., Kervadec, A., Pineda, S., Kalvakuri, S., Alayari, N. N., Foco, L., Pramstaller, P. P., Ocorr, K., Rossini, A., Cammarato, A., Colas, A. R., Hicks, A. A. and Bodmer, R. (2020). Silencing of CCR4-NOT complex subunits affect heart structure and function. Dis Model Mech. PubMed ID: 32471864
Genome wide association studies (GWAS) have identified variants that associate with QT-interval length. Three of the strongest associating variants (SNPs) are located in the putative promotor region of CNOT1, a gene encoding the central subunit of CCR4-NOT, a multi-functional, conserved complex regulating gene expression and mRNA stability and turnover. The minimum fragment of the CNOT1 promoter containing all three variants was isolated from individuals homozygous for the QT-risk alleles and it was then demonstrated that the haplotype associating with longer QT-interval caused reduced reporter expression in a cardiac cell line, suggesting that reduced CNOT1 expression may contribute to abnormal QT-intervals. Systematic siRNA-mediated knockdown of CCR4-NOT components in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) revealed that silencing CNOT1 and other CCR4-CNOT genes reduced their proliferative capacity. Silencing CNOT7 also shortened action potential duration. Furthermore, cardiac-specific knockdown of Drosophila orthologs of CCR4-NOT genes, CNOT1/not1 and CNOT7/8/pop2, in vivo, was either lethal or resulted in dilated cardiomyopathy, reduced contractility, or a propensity for arrhythmia. Silencing CNOT2/not2, CNOT4/not4 and CNOT6/6L/twin also affected cardiac chamber size and contractility. Developmental studies suggested that CNOT1/not1 and CNOT7/8/pop2 are required during cardiac remodeling from larval to adult stages. In sum, this study has demonstrated how disease associated genes identified by GWAS can be investigated, by combining human cardiomyocyte cell-based and whole organism in vivo heart models. These results also suggest a potential link of CNOT1 and CNOT7/8 to QT alterations and further establish a critical role of the CCR4-NOT complex in heart development and function.
Gonzalez, A. E. and Wang, X. (2020). Drosophila VCP/p97 Mediates Dynein-Dependent Retrograde Mitochondrial Motility in Axons. Front Cell Dev Biol 8: 256. PubMed ID: 32373611
Valosin-containing protein (VCP), also called p97, is an evolutionarily conserved and ubiquitously expressed ATPase with diverse cellular functions. Dominant mutations in VCP are found in a late-onset multisystem degenerative proteinopathy. The neurological manifestations of the disorder include frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In these patients, long motor neuron axons could be particularly susceptible to defects in axonal transport. However, whether VCP has a physiological function in maintaining axonal transport and whether this role is impaired by disease-causing mutations remains elusive. By employing live-imaging methods in Drosophila larval axons and performing genetic interaction experiments, this study discovered that VCP regulates the axonal transport of mitochondria. Downregulation of VCP enhances the retrograde transport of mitochondria and reduces the density of mitochondria in larval axons. This unidirectional motility phenotype is rescued by removing one copy of the retrograde motor dynein heavy chain (DHC), or elevating Miro which facilitates anterograde mitochondrial movement by interacting with the anterograde motor kinesin heavy chain (KHC). Importantly, Miro upregulation also significantly improves ATP production of VCP mutant larvae. Human VCP pathogenic mutations were investigated in the fly system. Expressing these mutations affects mitochondrial transport in the same way as knocking down VCP. These results reveal a new role of VCP in mediating axonal mitochondrial transport, and provide evidence implicating impaired mitochondrial motility in the pathophysiology of VCP-relevant neurodegenerative diseases.
Gangwani, K., Snigdha, K. and Kango-Singh, M. (2020). Tep1 Regulates Yki Activity in Neural Stem Cells in Drosophila Glioma Model. Front Cell Dev Biol 8: 306. PubMed ID: 32457905
Glioblastoma Multiforme (GBM) is the most common form of malignant brain tumor with poor prognosis. Amplification of Epidermal Growth Factor Receptor (EGFR), and mutations leading to activation of Phosphatidyl-Inositol-3 Kinase (PI3K) pathway are commonly associated with GBM. Using a previously published Drosophila glioma model generated by coactivation of PI3K and EGFR pathways [by downregulation of Pten and overexpression of oncogenic Ras] in glial cells, this study showed that the Drosophila Tep1 gene (ortholog of human CD109) regulates Yki (the Drosophila ortholog of human YAP/TAZ) via an evolutionarily conserved mechanism. Oncogenic signaling by the YAP/TAZ pathway occurs in cells that acquire CD109 expression in response to the inflammatory environment induced by radiation in clinically relevant models. Further, downregulation of Tep1 caused a reduction in Yki activity and reduced glioma growth. A key function of Yki in larval CNS is stem cell renewal and formation of neuroblasts. Other reports suggest different upstream regulators of Yki activity in the optic lobe versus the central brain regions of the larval CNS. It was hypothesized that Tep1 interacts with the Hippo pathway effector Yki to regulate neuroblast numbers. Tests were performed to see whether Tep1 acts through Yki to affect glioma growth and if in normal cells Tep1 affects neuroblast number and proliferation. These data suggests that Tep1 affects Yki mediated stem cell renewal in glioma, as reduction of Tep significantly decreases the number of neuroblasts in glioma. Thus, this study identifies Tep1-Yki interaction in the larval CNS that plays a key role in glioma growth and progression.
Hill, C. S., Sreedharan, J., Loreto, A., Menon, D. K. and Coleman, M. P. (2020). Loss of highwire Protects Against the Deleterious Effects of Traumatic Brain Injury in Drosophila Melanogaster. Front Neurol 11: 401. PubMed ID: 32477254
Traumatic brain injury is a major global cause of death and disability. Axonal injury is a major underlying mechanism of TBI and could represent a major therapeutic target. This study provides evidence that targeting the axonal death pathway known as Wallerian degeneration improves outcome in a Drosophila melanogaster model of high impact trauma. This cell-autonomous neurodegenerative pathway is initiated following axon injury, and in Drosophila, involves activity of the E3 ubiquitin ligase highwire. Loss-of-function mutation in the highwire gene rescues deleterious effects of a traumatic injury, including-improved functional outcomes, lifespan, survival of dopaminergic neurons, and retention of synaptic proteins. This data suggests that highwire represents a potential therapeutic target in traumatic injury.
Han, H., Tan, J., Wang, R., Wan, H., He, Y., Yan, X., Guo, J., Gao, Q., Li, J., Shang, S., Chen, F., Tian, R., Liu, W., Liao, L., Tang, B. and Zhang, Z. (2020). PINK1 phosphorylates Drp1(S616) to regulate mitophagy-independent mitochondrial dynamics. EMBO Rep: e48686. PubMed ID: 32484300
Impairment of PINK1/parkin-mediated mitophagy is currently proposed to be the molecular basis of mitochondrial abnormality in Parkinson's disease (PD). This study demonstrates that PINK1 directly phosphorylates Drp1 on S616. Drp1(S616) phosphorylation is significantly reduced in cells and mouse tissues deficient for PINK1, but unaffected by parkin inactivation. PINK1-mediated mitochondrial fission is Drp1(S616) phosphorylation dependent. Overexpression of either wild-type Drp1 or of the phosphomimetic mutant Drp1(S616D), but not a dephosphorylation-mimic mutant Drp1(S616A), rescues PINK1 deficiency-associated phenotypes in Drosophila. Moreover, Drp1 restores PINK1-dependent mitochondrial fission in ATG5-null cells and ATG7-null Drosophila. Reduced Drp1(S616) phosphorylation is detected in fibroblasts derived from 4 PD patients harboring PINK1 mutations and in 4 out of 7 sporadic PD cases. Taken together, this study has identified Drp1 as a substrate of PINK1 and a novel mechanism how PINK1 regulates mitochondrial fission independent of parkin and autophagy. These results further link impaired PINK1-mediated Drp1(S616) phosphorylation with the pathogenesis of both familial and sporadic PD.

Thursday, June 25th - Signaling

Hwang, J. H., Vuong, L. T. and Choi, K. W. (2020). Crumbs, Galla and Xpd are required for kinesin-5 regulation in mitosis and organ growth in Drosophila. J Cell Sci. PubMed ID: 32501288
Xeroderma Pigmentosum D (XPD) is a multi-function protein involved in transcription, DNA repair, and chromosome segregation. In Drosophila, Xpd interacts with Crumbs (Crb) and Galla to regulate mitosis during embryogenesis. It is unknown how these proteins are linked to mitosis. This study shows that Crb, Galla-2 and Xpd regulate nuclear division in syncytial embryo by interacting with Klp61F, the Drosophila mitotic kinesin-5 associated with bipolar spindles. Crb, Galla-2 and Xpd physically interact with Klp61F and co-localize to mitotic spindles. Knockdown of any of these proteins results in similar mitotic defects. These phenotypes are restored by overexpressing Klp61F, suggesting that Klp61F is a major effector. Mitotic defects of galla-2 RNAi are suppressed by Xpd overexpression but not vice versa Depletion of Crb, Galla-2 or Xpd results in a reduction of Klp61F levels. Reducing proteasome function restores Klp61F levels and suppress mitotic defects caused by knockdown of Crb, Galla-2 or Xpd. Further, eye growth is regulated by Xpd and Klp61F. Hence, this study proposes that Crb, Galla-2 and Xpd interact to maintain the level of Klp61F during mitosis and organ growth.
DeAngelis, M. W., McGhie, E. W., Coolon, J. D. and Johnson, R. I. (2020). Mask, a component of the Hippo pathway, is required for Drosophila eye morphogenesis. Dev Biol. PubMed ID: 32464117
Hippo signaling is an important regulator of tissue size, but it also has a lesser-known role in tissue morphogenesis. This study used the Drosophila pupal eye to explore the role of the Hippo effector Yki and its cofactor Mask in morphogenesis. Mask is required for the correct distribution and accumulation of adherens junctions and appropriate organization of the cytoskeleton. Accordingly, disrupting mask expression led to severe mis-patterning and similar defects were observed when yki was reduced or in response to ectopic wts. Further, the patterning defects generated by reducing mask expression were modified by Hippo pathway activity. RNA-sequencing revealed a requirement for Mask for appropriate expression of numerous genes during eye morphogenesis. These included genes implicated in cell adhesion and cytoskeletal organization, a comprehensive set of genes that promote cell survival, and numerous signal transduction genes. To validate the transcriptome analyses, two loci are considered that were modified by Mask activity: FER and Vinc, which have established roles in regulating adhesion. Modulating the expression of either locus modified mask mis-patterning and adhesion phenotypes. Further, expression of FER and Vinc was modified by Yki. It is well-established that the Hippo pathway is responsive to changes in cell adhesion and the cytoskeleton, but these data indicate that Hippo signaling also regulates these structures.
Chen, Y., Sun, T., Bi, Z., Ni, J. Q., Pastor-Pareja, J. C. and Javid, B. (2020). Premature termination codon readthrough in Drosophila varies in a developmental and tissue-specific manner. Sci Rep 10(1): 8485. PubMed ID: 32444687
Despite their essential function in terminating translation, readthrough of stop codons occurs more frequently than previously supposed. However, little is known about the regulation of stop codon readthrough by anatomical site and over the life cycle of animals. This study developed a set of reporters to measure readthrough in Drosophila melanogaster. A focused RNAi screen in whole animals identified upf1 as a mediator of readthrough, suggesting that the stop codons in the reporters were recognized as premature termination codons (PTCs). Readthrough rates of PTCs varied significantly throughout the life cycle of flies, being highest in older adult flies. Furthermore, readthrough rates varied dramatically by tissue and, intriguingly, were highest in fly brains, specifically neurons and not glia. This was not due to differences in reporter abundance or nonsense-mediated mRNA decay (NMD) surveillance between these tissues. Readthrough rates also varied within neurons, with cholinergic neurons having highest readthrough compared with lowest readthrough rates in dopaminergic neurons. Overall, these data reveal temporal and spatial variation of PTC-mediated readthrough in animals, and suggest that readthrough may be a potential rescue mechanism for PTC-harboring transcripts when the NMD surveillance pathway is inhibited.
Bruzzone, L., Arguelles, C., Sanial, M., Miled, S., Alvisi, G., Goncalves-Antunes, M., Qasrawi, F., Holmgren, R. A., Smibert, C. A., Lipshitz, H. D., Boccaccio, G. L., Plessis, A. and Becam, I. (2020). Regulation of the RNA-binding protein Smaug by the GPCR Smoothened via the kinase Fused. EMBO Rep: e48425. PubMed ID: 32383557
From fly to mammals, the Smaug/Samd4 family of prion-like RNA-binding proteins control gene expression by destabilizing and/or repressing the translation of numerous target transcripts. However, the regulation of its activity remains poorly understood. This study shows that Smaug's protein levels and mRNA repressive activity are downregulated by Hedgehog signaling in tissue culture cells. These effects rely on the interaction of Smaug with the G-protein coupled receptor Smoothened, which promotes the phosphorylation of Smaug by recruiting the kinase Fused. The activation of Fused and its binding to Smaug are sufficient to suppress its ability to form cytosolic bodies and to antagonize its negative effects on endogenous targets. Importantly, this study demonstrates in vivo that HH reduces the levels of smaug mRNA and increases the level of several mRNAs downregulated by Smaug. Finally, this study shows that Smaug acts as a positive regulator of Hedgehog signaling during wing morphogenesis. These data constitute the first evidence for a post-translational regulation of Smaug and reveal that the fate of several mRNAs bound to Smaug is modulated by a major signaling pathway.
Chen, Y., Kotian, N., Aranjuez, G., Chen, L., Messer, C. L., Burtscher, A., Sawant, K., Ramel, D., Wang, X. and McDonald, J. A. (2020). Protein phosphatase 1 activity controls a balance between collective and single cell modes of migration. Elife 9. PubMed ID: 32369438
Collective cell migration is central to many developmental and pathological processes. However, the mechanisms that keep cell collectives together and coordinate movement of multiple cells are poorly understood. Using the Drosophila border cell migration model, this study finds that Protein phosphatase 1 (Pp1; Drosophila Pp1α-96A, Pp1-87B, Pp1-13C and Flapwing) activity controls collective cell cohesion and migration. Inhibition of Pp1 causes border cells to round up, dissociate, and move as single cells with altered motility. Evidence is presented that Pp1 promotes proper levels of cadherin-catenin complex proteins at cell-cell junctions within the cluster to keep border cells together. Pp1 further restricts actomyosin contractility to the cluster periphery rather than at individual internal border cell contacts. The myosin phosphatase Pp1 complex, which inhibits non-muscle myosin-II (Myo-II/Zipper) activity, coordinates border cell shape and cluster cohesion. Given the high conservation of Pp1 complexes, this study identifies Pp1 as a major regulator of collective versus single cell migration.
Cannac, F., Qi, C., Falschlunger, J., Hausmann, G., Basler, K. and Korkhov, V. M. (2020). Cryo-EM structure of the Hedgehog release protein Dispatched. Sci Adv 6(16): eaay7928. PubMed ID: 32494603
The Hedgehog (Hh) signaling pathway controls embryonic development and adult tissue homeostasis in multicellular organisms. In Drosophila melanogaster, the pathway is primed by secretion of a dually lipid-modified morphogen, Hh, a process dependent on a membrane-integral protein Dispatched. Although Dispatched is a critical component of the pathway, the structural basis of its activity has, so far, not been described. This study describes a cryo-electron microscopy structure of the D. melanogaster Dispatched at 3.2-Å resolution. The ectodomains of Dispatched adopt an open conformation suggestive of a receptor-chaperone role. A three-dimensional reconstruction of Dispatched bound to Hh confirms the ability of Dispatched to bind Hh but using a unique mode distinct from those previously observed in structures of Hh complexes. The structure may represent the state of the complex that precedes shedding of Hh from the surface of the morphogen-releasing cell.

Wednesday, June 24th - Enhancers and gene regulation

Soluri, I. V., Zumerling, L. M., Payan Parra, O. A., Clark, E. G. and Blythe, S. A. (2020). Zygotic pioneer factor activity of Odd-paired/Zic is necessary for late function of the Drosophila segmentation network. Elife 9. PubMed ID: 32347792
Because chromatin determines whether information encoded in DNA is accessible to transcription factors, dynamic chromatin states in development may constrain how gene regulatory networks impart embryonic pattern. To determine the interplay between chromatin states and regulatory network function, Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was performed on Drosophila embryos during the establishment of the segmentation network, comparing wild-type and mutant embryos in which all graded maternal patterning inputs are eliminated. While during the period between zygotic genome activation and gastrulation many regions maintain stable accessibility, cis-regulatory modules (CRMs) within the network undergo extensive patterning-dependent changes in accessibility. A component of the network, Odd-paired (opa), is necessary for pioneering accessibility of late segmentation network CRMs. opa-driven changes in accessibility are accompanied by equivalent changes in gene expression. Interfering with the timing of opa activity impacts the proper patterning of expression. These results indicate that dynamic systems for chromatin regulation directly impact the reading of embryonic patterning information.
Mouawad, R., Himadewi, P., Kadiyala, D. and Arnosti, D. N. (2020). Selective repression of the Drosophila cyclin B promoter by retinoblastoma and E2F proteins. Biochim Biophys Acta Gene Regul Mech 1863(7): 194549. PubMed ID: 32275964
The Cyclin B1 gene encodes a G2/M cyclin that is deregulated in various human cancers, however, the transcriptional regulation of this gene is incompletely understood. The E2F and retinoblastoma family of proteins are involved in this gene's regulation, but there is disagreement on which of the E2F and retinoblastoma proteins interact with the promoter to regulate this gene. This study dissected the promoter region of the Drosophila CycB gene and studied the role of Rbf and E2F factors in its regulation. This gene exhibits remarkable features that distinguish it from G1/S regulated promoters, such as PCNA. The promoter is comprised of modular elements with dedicated repressor and activator functions, including a segment spanning the first intron that interferes with a 5' activator element. A highly active minimal promoter (-464, +100) is repressed by the Rbf1 retinoblastoma protein, but much more potently repressed by the Rbf2 protein, which has been linked in other studies to control of cell growth genes. Unlike many other cell-cycle genes, which are activated by E2F1 and repressed by E2F2, CycB is potently activated by E2F2, and repressed by E2F1. Although the bulk of Rbf binding is associated with a region 5' of the core promoter, E2F and retinoblastoma proteins functionally interact with the basal promoter region, in part through a conserved E2F site at -80 bp. The specific regulatory requirements of this late cell cycle promoter appear to be linked to the unique activities of E2F and retinoblastoma family members acting on a complex cis-regulatory circuit.
Miller, S. W. and Posakony, J. W. (2020). Disparate expression specificities coded by a shared Hox-C enhancer. Elife 9. PubMed ID: 32342858
Can a single regulatory sequence be shared by two genes undergoing functional divergence? This study describes a single promiscuous enhancer within the Drosophila Antennapedia Complex, EO053, that directs aspects of the expression of two adjacent genes, pb (a Hox2 ortholog) and zen2 (a divergent Hox3 paralog), with disparate spatial and temporal expression patterns. It was not possible to separate the pb-like and zen2-like specificities within EO053, and sequences affecting both expression patterns were identified. Importantly, genomic deletion experiments demonstrate that EO053 cooperates with additional pb- and zen2-specific enhancers to regulate the mRNA expression of both genes. Sequence conservation of EO053 was examined within the Schizophora, and patterns of synteny between the Hox2 and Hox3 orthologs in Arthropods are consistent with a shared regulatory relationship extending prior to the Hox3/zen divergence. Thus, EO053 represents an example of two genes having evolved disparate outputs while utilizing this shared regulatory region.
Bravo Gonzalez-Blas, C., Quan, X. J., Duran-Romanma, R., Taskiran, II, Koldere, D., Davie, K., Christiaens, V., Makhzami, S., Hulselmans, G., de Waegeneer, M., Mauduit, D., Poovathingal, S., Aibar, S. and Aerts, S. (2020). Identification of genomic enhancers through spatial integration of single-cell transcriptomics and epigenomics. Mol Syst Biol 16(5): e9438. PubMed ID: 32431014
Single-cell technologies allow measuring chromatin accessibility and gene expression in each cell, but jointly utilizing both layers to map bona fide gene regulatory networks and enhancers remains challenging. This study generated independent single-cell RNA-seq and single-cell ATAC-seq atlases of the Drosophila eye-antennal disc and spatially integrated the data into a virtual latent space that mimics the organization of the 2D tissue using ScoMAP (Single-Cell Omics Mapping into spatial Axes using Pseudotime ordering). To validate spatially predicted enhancers, a large collection of enhancer-reporter lines was used and ~ 85% of enhancers were identified in which chromatin accessibility and enhancer activity are coupled. Next, enhancer-to-gene relationships in the virtual space was inferred, finding that genes are mostly regulated by multiple, often redundant, enhancers. Exploiting cell type-specific enhancers, cell type-specific effects of bulk-derived chromatin accessibility QTLs were deconvoluted. Finally, it was discovered that Prospero drives neuronal differentiation through the binding of a GGG motif. In summary, this study provides a comprehensive spatial characterization of gene regulation in a 2D tissue.
Terzioglu Kara, E., Kiral, F. R., Ozturk Colak, A. and Celik, A. (2020). Generation and characterization of inner photoreceptor-specific enhancer-trap lines using a novel piggyBac-Gal4 element in Drosophila. Arch Insect Biochem Physiol: e21675. PubMed ID: 32285519
The Drosophila inner photoreceptors R7 and R8 are responsible for color vision and their differentiation starts at the third instar larval stage. Only a handful of genes with R7 or R8-cell-specific expression are known. An enhancer-trap screen was performed using a novel piggyBac transposable element, pBGay, carrying a Gal4 sequence under the control of the P promoter to identify novel genes expressed specifically in R7 or R8 cells. From this screen, three lines were analyzed in detail: piggyBac(AC109) and piggyBac(AC783) are expressed in R8 cells and piggyBac(AC887) is expressed in R7 cells at the third instar larval stage and pupal stages. Molecular analysis showed that the piggyBac elements were inserted into the first intron of CG14160 and CG7985 genes and the second intron of unzipped. The expression pattern was demonstrated in the developing eye imaginal disc, pupal retina as well as the adult retina. The photoreceptor-specific expression of these genes is reported for the first time and it is proposed that these lines are useful tools for studying the development of the visual system.
Harris, R. E., Stinchfield, M. J., Nystrom, S. L., McKay, D. J. and Hariharan, I. K. (2020). Damage-responsive, maturity-silenced enhancers regulate multiple genes that direct regeneration in Drosophila. Elife 9. PubMed ID: 32490812
Like tissues of many organisms, Drosophila imaginal discs lose the ability to regenerate as they mature. This loss of regenerative capacity coincides with reduced damage-responsive expression of multiple genes needed for regeneration. Previous work has shown that two such genes, wg and Wnt6, are regulated by a single damage-responsive enhancer that becomes progressively inactivated via Polycomb-mediated silencing as discs mature. This study explored the generality of this mechanism and identified additional damage-responsive, maturity-silenced (DRMS) enhancers, some near genes known to be required for regeneration such as Mmp1, and others near genes that this study now shows to function in regeneration. Using a novel GAL4-independent ablation system two DRMS-associated genes were characterized, apontic (apt), which curtails regeneration and CG9752/asperous (aspr), which promotes it. This mechanism of suppressing regeneration by silencing damage-responsive enhancers at multiple loci can be partially overcome by reducing activity of the chromatin regulator extra sex combs (esc).

Wednesday, June 23rd - Methods

Kandul, N. P., Liu, J., Buchman, A., Gantz, V. M., Bier, E. and Akbari, O. S. (2019). Assessment of a split homing based gene drive for efficient knockout of multiple genes. G3 (Bethesda). PubMed ID: 31882406
Homing based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent disease transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate genes has not been thoroughly explored. To test this approach, a split-HGD was generated in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host-encoded gene, that was termed a gRNA-mediated effector (GME). This design enabled assessing homing and knockout efficiencies of two target genes simultaneously, and also the timing and tissue specificity of Cas9 expression on cleavage/homing rates was explored. Inclusion of a GME can result in high efficiency of disruption of both genes during super-Mendelian propagation of split-HGD. Furthermore, both genes were knocked out one generation earlier than expected indicating the robust somatic expression of Cas9 driven by Drosophila germline-limited promoters. The efficiency of 'shadow drive' generated by maternally deposited Cas9 protein and accumulation of drive-induced resistance alleles was assessed along multiple generations, and design principles of HGD was discussed that could mitigate the accumulation of resistance alleles while incorporating a GME.
Edwards, K. A., Hoppa, M. B. and Bosco, G. (2020). The Photoconvertible Fluorescent Probe, CaMPARI, Labels Active Neurons in Freely-Moving Intact Adult Fruit Flies. Front Neural Circuits 14: 22. PubMed ID: 32457580
Linking neural circuitry to behavior by mapping active neurons in vivo is a challenge. Calcium-modulated photoactivatable ratiometric integrator (CaMPARI) was engineered to overcome spatial and temporal challenges. CaMPARI is a photoconvertible protein that only converts from green to red fluorescence in the presence of high calcium concentration and 405 nm light. This allows the experimenter to precisely mark active neurons within defined temporal windows. The photoconversion can then be quantified by taking the ratio of the red fluorescence to the green. CaMPARI promises the ability to trace active neurons during a specific stimulus; however, CaMPARI's uses in adult Drosophila have been limited to photoconversion during fly immobilization. This study demonstrates a method that allows photoconversion of multiple freely-moving intact adult flies during a stimulus. Flies were placed in a dish with filter paper wet with acetic acid (pH = 2) or neutralized acetic acid (pH = 7) and exposed to photoconvertible light (60 mW) for 30 min (500 ms on, 200 ms off). Immediately following photoconversion, whole flies were fixed and imaged by confocal microscopy. The red:green ratio was quantified for the DC4 glomerulus, a bundle of neurons expressing Ir64a, an ionotropic receptor that senses acids in the Drosophila antennal lobe. Flies exposed to acetic acid showed 1.3-fold greater photoconversion than flies exposed to neutralized acetic acid. This finding was recapitulated using a more physiological stimulus of apple cider vinegar. These results indicate that CaMPARI can be used to label neurons in intact, freely-moving adult flies and will be useful for identifying the circuitry underlying complex behaviors.
Bosch, P. S., Pepperl, J. and Basler, K. (2020). Anchor Away - A Fast, Reliable and Reversible Technique To Inhibit Proteins in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 32217630
Several techniques have been developed to study specific gene function in loss of function situations. In Drosophila melanogaster, RNAi and the generation of mutant clones are widely used. However, both techniques have the limitation that there is a significant time lag before gene function is abolished. Given the relatively rapid development of Drosophila, such perdurance is a serious impediment to study gene function. This study describes the adaptation of the anchor-away technique for use in Drosophila. Anchor-away was originally developed in yeast to quickly and efficiently abrogate the function of nuclear proteins by sequestering - anchoring - them away in a different cellular compartment. The required components are present in the cells, and the system is triggered by the addition of rapamycin, resulting in a rapid generation of a loss-of-function situation. This study provides proof of principle for the system by producing loss-of-function situations for two nuclear proteins - Pygopus and Brinker. The system allows studying of the requirement of any protein during any time window, and at the same time circumvents difficulties, such as off-target effects or variable phenotypes, which are inherent in other techniques, for example RNAi.
Grimes, L., Griffiths, J., Pasqualetto, G., Brancale, A., Kemp, P. J., Young, M. T. and van der Goes van Naters, W. (2020). Drosophila taste neurons as an agonist-screening platform for P2X receptors. Sci Rep 10(1): 8292. PubMed ID: 32427920
The P2X receptor family of ATP-gated cation channels are attractive drug targets for pain and inflammatory disease, but no subtype-selective agonists, and few partially selective agonists have been described to date. As proof-of-concept for the discovery of novel P2X receptor agonists, this study demonstrates the use of Drosophila taste neurons heterologously expressing rat P2X2 receptors as a screening platform. Wild-type rat P2X2 expressed in Drosophila is fully functional (ATP EC(50) 8.7 μM), and that screening of small (2 μl) volumes of a library of 80 adenosine nucleotide analogues is rapid and straightforward. Agonist potency and specificity profiles were determined for rat P2X2 receptors; triphosphate-bearing analogues display broad activity, tolerating a number of substitutions, and diphosphate and monophosphate analogues display very little activity. While several ATP analogues gave responses of similar magnitude to ATP, including the previously identified agonists ATPγS and ATPαS, a novel agonist, the synthetic analogue 2-fluoro-ATP, was identified and its agonist activity on rat P2X2 receptors expressed in human cells was confirmed. These data validate the Drosophila platform as a useful tool for the analysis of agonist structure-activity relationships, and for the screening and discovery of novel P2X receptor agonists.
Luan, H., Kuzin, A., Odenwald, W. F. and White, B. H. (2020). Cre-assisted fine-mapping of neural circuits using orthogonal split inteins. Elife 9. PubMed ID: 32286225
Existing genetic methods of neuronal targeting do not routinely achieve the resolution required for mapping brain circuits. New approaches are thus necessary. This study introduces a method for refined neuronal targeting that can be applied iteratively. Restriction achieved at the first step can be further refined in a second step, if necessary. The method relies on first isolating neurons within a targeted group (i.e. Gal4 pattern) according to their developmental lineages, and then intersectionally limiting the number of lineages by selecting only those in which two distinct neuroblast enhancers are active. The neuroblast enhancers drive expression of split Cre recombinase fragments. These are fused to non-interacting pairs of split inteins, which ensure reconstitution of active Cre when all fragments are expressed in the same neuroblast. Active Cre renders all neuroblast-derived cells in a lineage permissive for Gal4 activity. How this system can facilitate neural circuit-mapping in Drosophila is demonstrated.
Chen, H. M., Yao, X., Ren, Q., Chang, C. C., Liu, L. Y., Miyares, R. L. and Lee, T. (2020). Enhanced Golic+: highly effective CRISPR gene targeting and transgene HACKing in Drosophila. Development 147(11). PubMed ID: 32467238
Gene targeting is an incredibly valuable technique. Sometimes, however, it can also be extremely challenging for various intrinsic reasons (e.g. low target accessibility or nature/extent of gene modification). To bypass these barriers, a transgene-based system was designed in Drosophila that increases the number of independent gene targeting events while at the same time enriching for correctly targeted progeny. Unfortunately, with particularly challenging gene targeting experiments, the original design yielded numerous false positives. This study delivers a much-improved technique, named Enhanced Golic+ (E-Golic+). E-Golic+ incorporates genetic modifications to tighten lethality-based selection while simultaneously boosting efficiency. With E-Golic+, previously unattainable gene targeting was achieved. Additionally, an E-Golic+-based, high-efficiency genetic pipeline was built for transgene swapping. Its utility was demonstrated by transforming GAL4 enhancer-trap lines into tissue-specific Cas9-expressing lines. Given the superior efficiency, specificity and scalability, E-Golic+ promises to expedite development of additional sophisticated genetic/genomic tools in Drosophila.

Monday June 22nd - Adult physiology

Garcia, M. J., Littler, A. S., Sriram, A. and Teets, N. M. (2020). Distinct cold tolerance traits independently vary across genotypes in Drosophila melanogaster. Evolution. PubMed ID: 32463118
Cold tolerance, the ability to cope with low temperature stress, is a critical adaptation in thermally variable environments. An individual's cold tolerance comprises several traits including minimum temperatures for growth and activity, ability to survive severe cold, and ability to resume normal function after cold subsides. Across species, these traits are correlated, suggesting they were shaped by shared evolutionary processes or possibly share physiological mechanisms. However, the extent to which cold tolerance traits and their associated mechanisms covary within populations has not been assessed. This study measured five cold tolerance traits-critical thermal minimum, chill coma recovery, short- and long-term cold tolerance, and cold-induced changes in locomotor behavior-along with cold-induced expression of two genes with possible roles in cold tolerance (heat shock protein 70 and frost)-across 12 lines of Drosophila melanogaster derived from a single population. Significant genetic variation was observed in all traits, but few were correlated across genotypes, and these correlations were sex-specific. Further, cold-induced gene expression varied by genotype, but there was no evidence supporting the hypothesis that cold-hardy lines would have either higher baseline expression or induction of stress genes. These results suggest cold tolerance traits possess unique mechanisms and have the capacity to evolve independently.
El-Saadi, M. I., Ritchie, M. W., Davis, H. E. and MacMillan, H. A. (2020). Warm periods in repeated cold stresses protect Drosophila against ionoregulatory collapse, chilling injury, and reproductive deficits. J Insect Physiol 123: 104055. PubMed ID: 32380094
During cold stress, chill susceptible insects like Drosophila melanogaster suffer from a loss of ion and water balance, and the current model of recovery from chilling posits that re-establishment of ion homeostasis begins upon return to a warm environment, but that it takes minutes to hours for an insect to fully restore homeostasis. Following this ionoregulatory model of chill coma recovery, it is predicted that the longer the duration of the warm periods between cold stresses, the better a fly will endure a subsequent chill coma event and the more likely they will be to survive. It was also predicted, however, that this recovery may lead to reduced fecundity, possibly due to allocation of energy reserves away from reproduction. In this study, female D.melanogaster were treated to a long continuous cold stress (25 h at 0 °C), or experienced the same total time in the cold with repeated short (15 min), or long (120 min) breaks at 22 °C. Warm periods in general improved survival outcomes, and individuals that recovered for more time in between cold periods had significantly lower rates of injury, faster recovery from chill coma, and produced greater, rather than fewer, offspring. These improvements in chill tolerance were associated with mitigation of ionoregulatory collapse, as flies that experienced either short or long warm periods better maintained low hemolymph [K(+)]. Thus, warm periods that interrupt cold periods improve cold tolerance and fertility in D. melanogaster females relative to a single sustained cold stress, potentially because this time allows for recovery of ion and water homeostasis.
Ma, C., Mirth, C. K., Hall, M. D. and Piper, M. D. W. (2020). Amino acid quality modifies the quantitative availability of protein for reproduction in Drosophila melanogaster. J Insect Physiol: 104050. PubMed ID: 32229142
Diet composition, especially the relative abundance of key macronutrients, is well known to affect animal wellbeing by changing reproductive output, metabolism and length of life. However, less attention has been paid to the ways the quality of these nutrients modify these macronutrient interactions. Nutritional Geometry can be used to model the effects of multiple dietary components on life-history traits and to compare these responses when diet quality is varied. Previous studies have shown that dietary protein quality can be increased for egg production in Drosophila melanogaster by matching the dietary amino acid proportions to the balance of amino acids used by the sum of proteins in the fly's in silico translated exome. This study shows that dietary protein quality dramatically alters the effect of protein quantity on female reproduction across a broad range of diets varying in both protein and carbohydrate concentrations. These data show that when sources of ingredients vary, their relative value to the consumer can vastly differ and yield very different physiological outcomes. Such variations could be particularly important for meta analyses that look to draw generalisable conclusions from diverse studies.
Pocas, G. M., Crosbie, A. E. and Mirth, C. K. (2020). When does diet matter? The roles of larval and adult nutrition in regulating adult size traits in Drosophila melanogaster. J Insect Physiol: 104051. PubMed ID: 32229143
Adult body size is determined by the quality and quantity of nutrients available to animals. In insects, nutrition affects adult size primarily during the nymphal or larval stages. However, measures of adult size like body weight are likely to also change with adult nutrition. This study sought to explore the roles of nutrition throughout the life cycle on adult body weight and the size of two appendages, the wing and the femur, in the fruit fly Drosophila melanogaster. Nutrition was manipulated in two ways: by varying the protein to carbohydrate content of the diet, called macronutrient restriction, and by changing the caloric density of the diet, termed caloric restriction. A fully factorial design was employed to manipulate both the larval and adult diets for both diet types. Manipulating the larval diet had greater impacts on all measures of adult size. Further, macronutrient restriction was more detrimental to adult size than caloric restriction. For adult body weight, a rich adult diet mitigated the negative effects of poor larval nutrition for both types of diets. In contrast, small wing and femur size caused by poor larval diet could not be increased with the adult diet. Taken together, these results suggest that appendage size is fixed by the larval diet, while those related to body composition remain sensitive to adult diet. Further, these studies provide a foundation for understanding how the nutritional environment of juveniles affects how adults respond to diet.
Matsushita, R. and Nishimura, T. (2020). Trehalose metabolism confers developmental robustness and stability in Drosophila by regulating glucose homeostasis. Commun Biol 3(1): 170. PubMed ID: 32265497
Organisms have evolved molecular mechanisms to ensure consistent and invariant phenotypes in the face of environmental fluctuations. Developmental homeostasis is determined by two factors: robustness, which buffers against environmental variations; and developmental stability, which buffers against intrinsic random variations. However, understanding of these noise-buffering mechanisms remains incomplete. This study has shown that appropriate glycemic control confers developmental homeostasis in the fruit fly Drosophila. Circulating glucose levels are buffered by trehalose metabolism, which acts as a glucose sink in circulation. Furthermore, mutations in trehalose synthesis enzyme (Tps1) increased the among-individual and within-individual variations in wing size. Whereas wild-type flies were largely resistant to changes in dietary carbohydrate and protein levels, Tps1 mutants experienced significant disruptions in developmental homeostasis in response to dietary stress. These results demonstrate that glucose homeostasis against dietary stress is crucial for developmental homeostasis.
Barretto, E. C., Polan, D. M., Beevor-Potts, A. N., Lee, B. and Grewal, S. S. (2020). Tolerance to Hypoxia Is Promoted by FOXO Regulation of the Innate Immunity Transcription Factor NF-κB/Relish in Drosophila. Genetics. PubMed ID: 32513813
Exposure of tissues and organs to low oxygen (hypoxia) occurs in both physiological and pathological conditions in animals. Under these conditions, organisms have to adapt their physiology to ensure proper functioning and survival. This study defined a role for the transcription factor FOXO as a mediator of hypoxia tolerance in Drosophila. Upon hypoxia exposure, FOXO transcriptional activity is rapidly induced in both larvae and adults. Moreover, foxo mutant animals show misregulated glucose metabolism in low oxygen and subsequently exhibit reduced hypoxia survival. The innate immune transcription factor, NF-κB/Relish, was identified as a key FOXO target in the control of hypoxia tolerance. Expression of Relish and its target genes are increased in a FOXO-dependent manner in hypoxia, and relish mutant animals show reduced survival in hypoxia. Together, these data indicate that FOXO is a hypoxia inducible factor that mediates tolerance to low oxygen by inducing immune-like responses.

Friday, June 19th - Disease Models

Takats, S., Levay, L., Boda, A., Toth, S., Simon-Vecsei, Z., Rubics, A., Varga, A., Lippai, M., Lorincz, P., Glatz, G. and Juhasz, G. (2020). The Warburg Micro Syndrome-associated Rab3GAP-Rab18 module promotes autolysosome maturation through the Vps34 Complex I. Febs j. PubMed ID: 32248620
Warburg micro syndrome (WMS) is a hereditary autosomal neuromuscular disorder in humans caused by mutations in Rab18, Rab3GAP1, or Rab3GAP2 genes. Rab3GAP1/2 forms a heterodimeric complex, which acts as a guanosine nucleotide exchange factor and activates Rab18. Although the genetic causes of WMS are known, it is still unclear whether loss of the Rab3GAP-Rab18 module affects neuronal or muscle cell physiology or both, and how. This work characterize a Rab3GAP2 mutant Drosophila line to establish a novel animal model for WMS. Similarly to symptoms of WMS, loss of Rab3GAP2 leads to highly decreased motility in Drosophila that becomes more serious with age. These mutant flies are defective for autophagic degradation in multiple tissues including fat cells and muscles. Loss of Rab3GAP-Rab18 module members leads to perturbed autolysosome morphology due to destabilization of Rab7-positive autophagosomal and late endosomal compartments and perturbation of lysosomal biosynthetic transport. Importantly, overexpression of UVRAG or loss of Atg14, two alternative subunits of the Vps34/PI3K (vacuole protein sorting 34/phosphatidylinositol 3-kinase) complexes in fat cells, mimics the autophagic phenotype of Rab3GAP-Rab18 module loss. This study finds that GTP-bound Rab18 binds to Atg6/Beclin1, a permanent subunit of Vps34 complexes. Finally, this study shows that Rab3GAP2 and Rab18 are present on autophagosomal and autolysosomal membranes and colocalize with Vps34 Complex I subunits. These data suggest that the Rab3GAP-Rab18 module regulates autolysosomal maturation through its interaction with the Vps34 Complex I, and perturbed autophagy due to loss of the Rab3GAP-Rab18 module may contribute to the development of WMS.
Xia, S. R., Wen, X. Y., Fan, X. L., Chen, X. R., Wei, Z. W., Li, Q. H. and Sun, L. (2020). Wnt2 overexpression protects against PINK1 mutant induced mitochondrial dysfunction and oxidative stress. Mol Med Rep 21(6): 2633-2641. PubMed ID: 32323790
The PTEN induced putative kinase 1 (PINK1) mutation is the second most common cause of autosomal recessive adolescent Parkinson's disease (PD). Furthermore, mitochondrial disorders and oxidative stress are important mechanisms in the pathogenesis of PD. Numerous members of the Wnt family have been found to be associated with neurodegenerative diseases. Therefore, the present study investigated the role of the Wnt2 gene in PINK1B9 transgenic flies, which is a PD model, and its underlying mechanism. It was identified that overexpression of Wnt2 reduced the abnormality rate of PD transgenic Drosophila and improved their flight ability, while other intervention groups had no significant effect. Furthermore, an increase in ATP concentration normalized mitochondrial morphology, and increased the mRNA expression levels of NADHubiquinone oxidoreductase chain 1 (ND1), ND42, ND75, succinate dehydrogenase complex subunits B, Cytochrome b and Cyclooxygenase 1, which are associated with Wnt2 overexpression. Moreover, overexpression of Wnt2 in PD transgenic Drosophila resulted in the downregulation of reactive oxygen species and malondialdehyde production, and increased manganese superoxide dismutase (MnSOD), while glutathione was not significantly affected. It was found that overexpression of Wnt2 did not alter the protein expression of betacatenin in PINK1B9 transgenic Drosophila, but did increase the expression levels of PPARG coactivator 1alpha (PGC1alpha) and forkhead box subgroup O (FOXO). Collectively, the present results indicated that the Wnt2 gene may have a protective effect on PD PINK1B9 transgenic Drosophila. Thus, it was speculated that the reduction of oxidative stress and the restoration of mitochondrial function via Wnt2 overexpression may be related to the PGC1alpha/FOXO/MnSOD signaling pathway in PINK1 mutant transgenic Drosophila.
Cortese, A., Zhu, Y., Rebelo, A. P.....Zhai, R. G. and Zuchner, S. (2020). Biallelic mutations in SORD cause a common and potentially treatable hereditary neuropathy with implications for diabetes. Nat Genet 52(5): 473-481. PubMed ID: 32367058
This study report biallelic mutations in the sorbitol dehydrogenase gene (SORD) as the most frequent recessive form of hereditary neuropathy. 45 individuals were identified from 38 families across multiple ancestries carrying the nonsense c.757delG (p.Ala253GlnfsTer27) variant in SORD, in either a homozygous or compound heterozygous state. SORD is an enzyme that converts sorbitol into fructose in the two-step polyol pathway previously implicated in diabetic neuropathy. In patient-derived fibroblasts, a complete loss of SORD protein and increased intracellular sorbitol were found. Furthermore, the serum fasting sorbitol levels in patients were dramatically increased. In Drosophila, loss of SORD orthologs caused synaptic degeneration and progressive motor impairment. Reducing the polyol influx by treatment with aldose reductase inhibitors normalized intracellular sorbitol levels in patient-derived fibroblasts and in Drosophila, and also dramatically ameliorated motor and eye phenotypes. Together, these findings establish a novel and potentially treatable cause of neuropathy and may contribute to a better understanding of the pathophysiology of diabetes.
Donnelly, K. M., DeLorenzo, O. R., Zaya, A. D., Pisano, G. E., Thu, W. M., Luo, L., Kopito, R. R. and Panning Pearce, M. M. (2020). Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses. Elife 9. PubMed ID: 32463364
>Emerging evidence supports the hypothesis that pathogenic protein aggregates associated with neurodegenerative diseases spread from cell to cell through the brain in a manner akin to infectious prions. This study shows that mutant huntingtin (mHtt) aggregates associated with Huntington disease transfer anterogradely from presynaptic to postsynaptic neurons in the adult Drosophila olfactory system. Trans-synaptic transmission of mHtt aggregates is inversely correlated with neuronal activity and blocked by inhibiting caspases in presynaptic neurons, implicating synaptic dysfunction and cell death in aggregate spreading. Remarkably, mHtt aggregate transmission across synapses requires the glial scavenger receptor Draper and involves a transient visit to the glial cytoplasm, indicating that phagocytic glia act as obligatory intermediates in aggregate spreading between synaptically-connected neurons. These findings expand understanding of phagocytic glia as double-edged players in neurodegeneration-by clearing neurotoxic protein aggregates, but also providing an opportunity for prion-like seeds to evade phagolysosomal degradation and propagate further in the brain.
Shiba-Fukushima, K., Inoshita, T., Sano, O., Iwata, H., Ishikawa, K. I., Okano, H., Akamatsu, W., Imai, Y. and Hattori, N. (2020). A Cell-Based High-Throughput Screening Identified Two Compounds that Enhance PINK1-Parkin Signaling. iScience 23(5): 101048. PubMed ID: 32335362
Early-onset Parkinson's disease-associated PINK1-Parkin signaling maintains mitochondrial health. Therapeutic approaches for enhancing PINK1-Parkin signaling present a potential strategy for treating various diseases caused by mitochondrial dysfunction. This study reports two chemical enhancers of PINK1-Parkin signaling, identified using a robust cell-based high-throughput screening system. These small molecules, T0466 and T0467, activate Parkin mitochondrial translocation in dopaminergic neurons and myoblasts at low doses that do not induce mitochondrial accumulation of PINK1. Moreover, both compounds reduce unfolded mitochondrial protein levels, presumably through enhanced PINK1-Parkin signaling. These molecules also mitigate the locomotion defect, reduced ATP production, and disturbed mitochondrial Ca(2+) response in the muscles along with the mitochondrial aggregation in dopaminergic neurons through reduced PINK1 activity in Drosophila. These results suggested that T0466 and T0467 may hold promise as therapeutic reagents in Parkinson's disease and related disorders.
Akasaka, T., Ocorr, K., Lin, L., Vogler, G., Bodmer, R. and Grossfeld, P. (2020). Overexpression of Kif1A in the Developing Drosophila Heart Causes Valvar and Contractility Defects: Implications for Human Congenital Heart Disease. J Cardiovasc Dev Dis 7(2). PubMed ID: 32498427
Left-sided congenital heart defects (CHDs) are among the most common forms of congenital heart disease, but a disease-causing gene has only been identified in a minority of cases. This study identified a candidate gene for CHDs, KIF1A, that was associated with a chromosomal balanced translocation t(2;8)(q37;p11) in a patient with left-sided heart and aortic valve defects. The breakpoint was in the 5' untranslated region of the KIF1A gene at 2q37, which suggested that the break affected the levels of Kif1A gene expression. Transgenic fly lines overexpressing Kif1A specifically in the heart muscle (or all muscles) caused diminished cardiac contractility, myofibrillar disorganization, and heart valve defects, whereas cardiac knockdown had no effect on heart structure or function. Overexpression of Kif1A also caused increased collagen IV deposition in the fibrous network that normally surrounds the fly heart. Kif1A overexpression in C2C12 myoblasts resulted in specific displacement of the F-actin fibers, probably through a direct interaction with G-actin. These results point to a Kif1A-mediated disruption of F-actin organization as a potential mechanism for the pathogenesis in at least some human CHDs.

Thursday Jun 18th - RNA

Leismann, J., Spagnuolo, M., Pradhan, M., Wacheul, L., Vu, M. A., Musheev, M., Mier, P., Andrade-Navarro, M. A., Graille, M., Niehrs, C., Lafontaine, D. L. and Roignant, J. Y. (2020). The 18S ribosomal RNA m(6) A methyltransferase Mettl5 is required for normal walking behavior in Drosophila. EMBO Rep: e49443. PubMed ID: 32350990
RNA modifications have recently emerged as an important layer of gene regulation. N6-methyladenosine (m(6) A) is the most prominent modification on eukaryotic messenger RNA and has also been found on noncoding RNA, including ribosomal and small nuclear RNA. Recently, several m(6) A methyltransferases were identified, uncovering the specificity of m(6) A deposition by structurally distinct enzymes. In order to discover additional m(6) A enzymes, this study performed an RNAi screen to deplete annotated orthologs of human methyltransferase-like proteins (METTLs) in Drosophila cells, and CG9666, the ortholog of human METTL5, was identified. CG9666 is required for specific deposition of m(6) A on 18S ribosomal RNA via direct interaction with the Drosophila ortholog of human TRMT112, CG12975. Depletion of CG9666 yields a subsequent loss of the 18S rRNA m(6) A modification, which lies in the vicinity of the ribosome decoding center; however, this does not compromise rRNA maturation. Instead, a loss of CG9666-mediated m(6) A impacts fly behavior, providing an underlying molecular mechanism for the reported human phenotype in intellectual disability. Thus, this work expands the repertoire of m(6) A methyltransferases, demonstrates the specialization of these enzymes, and further addresses the significance of ribosomal RNA modifications in gene expression and animal behavior.
Komarov, P. A., Sokolova, O., Akulenko, N., Brasset, E., Jensen, S. and Kalmykova, A. (2020). Epigenetic Requirements for Triggering Heterochromatinization and Piwi-Interacting RNA Production from Transgenes in the Drosophila Germline. Cells 9(4). PubMed ID: 32290057
Transgenes containing a fragment of the I retrotransposon represent a powerful model of piRNA cluster de novo formation in the Drosophila germline. This study revealed that the same transgenes located at different genomic loci form piRNA clusters with various capacity of small RNA production. Transgenic piRNA clusters are not established in piRNA pathway mutants. However, in the wild-type context, the endogenous ancestral I-related piRNAs heterochromatinize and convert the I-containing transgenes into piRNA-producing loci. This study addressed how the quantitative level of piRNAs influences the heterochromatinization and piRNA production. A minimal amount of maternal piRNAs from ancestral I-elements was shown to be sufficient to form the transgenic piRNA clusters. Supplemental piRNAs stemming from active I-element copies do not stimulate additional chromatin changes or piRNA production from transgenes. Therefore, chromatin changes and piRNA production are initiated by a minimum threshold level of complementary piRNAs, suggesting a selective advantage of prompt cell response to the lowest level of piRNAs. It is noteworthy that the weak piRNA clusters do not transform into strong ones after being targeted by abundant I-specific piRNAs, indicating the importance of the genomic context for piRNA cluster establishment. Analysis of ovarian transcription profiles suggests that regions facilitating convergent transcription favor the formation of transgenic piRNA clusters.
Li, Y., Zhang, X. M., Luan, M. W., Xing, J. F., Chen, J. and Xie, S. Q. (2020). Distribution Patterns of DNA N6-Methyladenosine Modification in Non-coding RNA Genes. Front Genet 11: 268. PubMed ID: 32265991
N6-methyladenosine (6mA) DNA modificatio played an important role in epigenetic regulation of gene expression. And the aberrational expression of non-coding genes, as important regular elements of gene expression, was related to many diseases. However, the distribution and potential functions of 6mA modification in non-coding RNA (ncRNA) genes are still unknown. This study analyzed the 6mA distribution of ncRNA genes and compared them with protein-coding genes in four species (Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens) using single-molecule real-time (SMRT) sequencing data. The results indicated that the consensus motifs of short nucleotides at 6mA location were highly conserved in four species, and the non-coding gene was less likely to be methylated compared with protein-coding gene. Especially, the 6mA-methylated lncRNA genes were expressed significant lower than these genes without methylation in A. thaliana (p = 3.295e-4), D. melanogaster (p = 3.439e-11), and H. sapiens (p = 9.087e-3) all four species. The detection and distribution profiling of 6mA modification in ncRNA regions from four species reveal that 6mA modifications may have effects on their expression level.
Borbolis, F., Rallis, J., Kanatouris, G., Kokla, N., Karamalegkos, A., Vasileiou, C., Vakaloglou, K. M., Diallinas, G., Stravopodis, D. J., Zervas, C. G. and Syntichaki, P. (2020). mRNA decapping is an evolutionarily conserved modulator of neuroendocrine signaling that controls development and ageing. Elife 9. PubMed ID: 32366357
Eukaryotic 5'-3' mRNA decay plays important roles during development and in response to stress, regulating gene expression post-transcriptionally. In Caenorhabditis elegans, deficiency of DCAP-1/DCP1, the essential co-factor of the major cytoplasmic mRNA decapping enzyme, impacts normal development, stress survival and ageing. This study shows that overexpression of dcap-1 in neurons of worms is sufficient to increase lifespan through the function of the insulin/IGF-like signaling and its effector DAF-16/FOXO transcription factor. Neuronal DCAP-1 affects basal levels of INS-7, an ageing-related insulin-like peptide, which acts in the intestine to determine lifespan. Short-lived dcap-1 mutants exhibit a neurosecretion-dependent upregulation of intestinal ins-7 transcription, and diminished nuclear localization of DAF-16/FOXO. Moreover, neuronal overexpression of DCP1 in Drosophila melanogaster confers longevity in adults, while neuronal DCP1 deficiency shortens lifespan and affects wing morphogenesis, cell non-autonomously. This genetic analysis in two model-organisms suggests a critical and conserved function of DCAP-1/DCP1 in developmental events and lifespan modulation.
Samuels, T. J., Arava, Y., Jarvelin, A. I., Robertson, F., Lee, J. Y., Yang, L., Yang, C. P., Lee, T., Ish-Horowicz, D. and Davis, I. (2020). Neuronal upregulation of Prospero protein is driven by alternative mRNA polyadenylation and Syncrip-mediated mRNA stabilisation. Biol Open. PubMed ID: 32205310
During Drosophila and vertebrate brain development, the conserved transcription factor Prospero/Prox1 is an important regulator of the transition between proliferation and differentiation. Prospero level is low in neural stem cells and their immediate progeny, but is upregulated in larval neurons and it is unknown how this process is controlled. This study used single molecule fluorescent in situ hybridisation to show that larval neurons selectively transcribe a long prospero mRNA isoform containing a 15 kb 3' untranslated region, which is bound in the brain by the conserved RNA-binding protein Syncrip/hnRNPQ. Syncrip binding increases the mRNA stability of the long prospero isoform, which allows an upregulation of Prospero protein production. Adult flies selectively lacking the long prospero isoform show abnormal behaviour that could result from impaired locomotor or neurological activity. These findings highlight a regulatory strategy involving alternative polyadenylation followed by differential post-transcriptional regulation.
Serrato-Capuchina, A., Wang, J., Earley, E., Peede, D., Isbell, K. and Matute, D. R. (2020). Paternally inherited P-element copy number affects the magnitude of hybrid dysgenesis in Drosophila simulans and D. melanogaster. Genome Biol Evol. PubMed ID: 32339225
Transposable elements (TEs) are repetitive regions of DNA that are able to self-replicate and reinsert themselves throughout host genomes. Since the discovery of TEs, a prevalent question has been whether increasing TE copy number has an effect on the fitness of their hosts. P-elements (PEs) in Drosophila are a well-studied TE that has strong phenotypic effects. When a female without PEs (M) is crossed to a male with them (P), the resulting females are often sterile, a phenomenon called hybrid dysgenesis (HD). This study used short and long-read sequencing to infer the number of PEs in the genomes of dozens of isofemale lines from two Drosophila species and measured whether the magnitude of HD was correlated with the number of PEs in the paternal genome. Consistent with previous reports, evidence was found for a positive correlation between the paternal PE copy number and the magnitude of HD in progeny from ♀M x ♂ P crosses for both species. Other crosses are not affected by the number of PE copies. This study also found that the correlation between the strength of HD and PE copy number differs between species which suggest there are genetic differences that might make some genomes more resilient to the potentially deleterious effects of TEs. These results suggest that PE copy number interacts with other factors in the genome and the environment to cause HD and that the importance of these interactions is species-specific.

Wednesday June 17 - Cytoskeleton and Junctions

Lamb, M. C., Anliker, K. K. and Tootle, T. L. (2020). Fascin regulates protrusions and delamination to mediate invasive, collective cell migration in vivo. Dev Dyn. PubMed ID: 32352613
The actin bundling protein Fascin is essential for developmental cell migrations and promotes cancer metastasis. In addition to bundling actin, Fascin has several actin-independent roles; how these other functions contribute to cell migration remains unclear. Border cell migration during Drosophila oogenesis provides an excellent model to study Fascin's various roles during invasive, collective cell migration. On-time border cell migration during Stage 9 requires Fascin (Drosophila Singed). Fascin functions not only within the migrating border cells, but also within the nurse cells, the substrate for this migration. Fascin genetically interacts with the actin elongation factor Enabled to promote on-time Stage 9 migration and overexpression of Enabled suppresses the defects seen with loss of Fascin. Loss of Fascin results in increased, shorter and mislocalized protrusions during migration. Additionally, loss of Fascin inhibits border cell delamination and increases E-Cadherin (Drosophila Shotgun) adhesions on both the border cell clusters and nurse cells. Overall, Fascin promotes on-time border cell migration during Stage 9 and contributes to multiple aspects of this invasive, collective cell migration, including both protrusion dynamics and delamination. These findings have implications beyond Drosophila, as border cell migration has emerged as a model to study mechanisms mediating cancer metastasis.
Del Castillo, U., Muller, H. J. and Gelfand, V. I. (2020). Kinetochore protein Spindly controls microtubule polarity in Drosophila axons. Proc Natl Acad Sci U S A 117(22): 12155-12163. PubMed ID: 32430325
Microtubule polarity in axons and dendrites defines the direction of intracellular transport in neurons. Axons contain arrays of uniformly polarized microtubules with plus-ends facing the tips of the processes (plus-end-out), while dendrites contain microtubules with a minus-end-out orientation. It has been shown that cytoplasmic dynein, targeted to cortical actin, removes minus-end-out microtubules from axons. This study has identified Spindly, a protein known for recruitment of dynein to kinetochores in mitosis, as a key factor required for dynein-dependent microtubule sorting in axons of Drosophila neurons. Depletion of Spindly affects polarity of axonal microtubules in vivo and in primary neuronal cultures. In addition to these defects, depletion of Spindly in neurons causes major collapse of axonal patterning in the third-instar larval brain as well as severe coordination impairment in adult flies. These defects can be fully rescued by full-length Spindly, but not by variants with mutations in its dynein-binding site. Biochemical analysis demonstrated that Spindly binds F-actin, suggesting that Spindly serves as a link between dynein and cortical actin in axons. Therefore, Spindly plays a critical role during neurodevelopment by mediating dynein-driven sorting of axonal microtubules (Del Castillo, 2020).
Pulido Companys, P., Norris, A. and Bischoff, M. (2020). Coordination of cytoskeletal dynamics and cell behaviour during Drosophila abdominal morphogenesis. J Cell Sci 133(6). PubMed ID: 32229579
During morphogenesis, cells exhibit various behaviours, such as migration and constriction, which need to be coordinated. How this is achieved remains elusive. During morphogenesis of the Drosophila adult abdominal epidermis, larval epithelial cells (LECs) migrate directedly before constricting apically and undergoing apoptosis. This study investigates the mechanisms underlying the transition from migration to constriction. LECs possess a pulsatile apical actomyosin network, and a change in network polarity correlates with behavioural change. Exploring the properties of the contractile network, it was found that cell contractility, as determined by myosin activity, has an impact on the behaviour of the network, as well as on cytoskeletal architecture and cell behaviour. Pulsed contractions occur only in cells with intermediate levels of contractility. Furthermore, increasing levels of the small Rho GTPase Rho1 disrupts pulsing, leading to cells that cycle between two states, characterised by a junctional cortical and an apicomedial actin network. These results highlight that behavioural change relies on tightly controlled cellular contractility. Moreover, constriction can occur without pulsing, raising questions why constricting cells pulse in some contexts but not in others.
Sherlekar, A., Mundhe, G., Richa, P., Dey, B., Sharma, S. and Rikhy, R. (2020). F-BAR domain protein Syndapin regulates actomyosin dynamics during apical cap remodeling in syncytial Drosophila embryos. J Cell Sci. PubMed ID: 32327556
Branched actin networks driven by Arp2/3 collaborate with actomyosin filaments in processes such as cell migration. The syncytial Drosophila blastoderm embryo also shows expansion of apical caps by Arp2/3 driven actin polymerization in interphase and buckling at contact edges by MyosinII to form furrows in metaphase. The role of Syndapin (Synd), an F-BAR domain containing protein in apical cap remodelling prior to furrow extension. synd depletion showed larger apical caps. STED super-resolution and TIRF microscopy showed long apical actin protrusions in caps in interphase and short protrusions in metaphase in control embryos. synd depletion led to sustained long protrusions even in metaphase. Loss of Arp2/3 function in synd mutants partly reverted defects in apical cap expansion and protrusion remodelling. MyosinII levels were decreased in synd mutants and MyosinII mutant embryos have been previously reported to have expanded caps. It is proposed that Syndapin function limits branching activity during cap expansion and affects MyosinII distribution in order to shift actin remodeling from apical cap expansion to favor lateral furrow extension.
Tang, Q., Rui, M., Bu, S., Wang, Y., Chew, L. Y. and Yu, F. (2020). A microtubule polymerase is required for microtubule orientation and dendrite pruning in Drosophila. EMBO J: e103549. PubMed ID: 32267553
Drosophila class IV ddaC neurons selectively prune all larval dendrites to refine the nervous system during metamorphosis. During dendrite pruning, severing of proximal dendrites is preceded by local microtubule (MT) disassembly. This study identified an unexpected role of Mini spindles (Msps), a conserved MT polymerase, in governing dendrite pruning. Msps associates with another MT-associated protein TACC, and both stabilize each other in ddaC neurons. Moreover, Msps and TACC are required to orient minus-end-out MTs in dendrites. It was further shown that the functions of msps in dendritic MT orientation and dendrite pruning are antagonized by the kinesin-13 MT depolymerase Klp10A. Excessive MT depolymerization, which is induced by pharmacological treatment and katanin overexpression, also perturbs dendritic MT orientation and dendrite pruning, phenocopying msps mutants. Thus, this study demonstrated that the MT polymerase Msps is required to form dendritic minus-end-out MTs and thereby promotes dendrite pruning in Drosophila sensory neurons.
Bauerly, E., Yi, K. and Gibson, M. C. (2020). Wampa is a dynein subunit required for axonemal assembly and male fertility in Drosophila. Dev Biol. PubMed ID: 32387369
In cilia and flagella, dyneins form complexes which give rise to the inner and outer axonemal arms. Defects in the dynein arms are the leading cause of primary ciliary dyskinesia (PCD), which is characterized by chronic respiratory infections, situs inversus, and sterility. While the pathological features associated with PCD are increasingly well characterized, many of the causative genetic lesions remain elusive. Using Drosophila, this study analyzed genetic requirements for wampa (wam; CG17083), a previously uncharacterized component of the outer dynein arm. While homozygous mutant animals are viable and display no morphological defects, loss of wam results in complete male sterility. Ultrastructural analysis further reveals that wam mutant spermatids lack the axonemal outer dynein arms, which leads to a complete loss of flagellar motility. In addition to a role in outer dynein arm formation, other novel microtubule-associated requirements were uncovered for wam during spermatogenesis, including the regulation of mitochondrial localization and the shaping of the nuclear head. Due to the conserved nature of dyneins, this study advances understanding of the pathology of PCD and the functional role of dyneins in axoneme formation and other aspects of spermatogenesis.

Tuesday, June 16th - Adult neural development and function

Turrel, O., Rabah, Y., Placais, P. Y., Goguel, V. and Preat, T. (2020). Drosophila middle-term memory: Amnesiac is required for PKA activation in the mushroom bodies, a function modulated by Neprilysin 1. J Neurosci. PubMed ID: 32303647
In Drosophila, the mushroom bodies (MB) constitute the central brain structure for olfactory associative memory. As in mammals, the cAMP/PKA pathway plays a key role in memory formation. In the MB, Rutabaga adenylate cyclase acts as a coincidence detector during associative conditioning to integrate calcium influx resulting from acetylcholine stimulation and G protein activation resulting from dopaminergic stimulation. Amnesiac encodes a secreted neuropeptide required in the MB for two phases of aversive olfactory memory. Previous sequence analysis has revealed strong homology with the mammalian pituitary adenylate cyclase-activating peptide (PACAP). This study examined whether amnesiac is involved in cAMP/PKA dynamics in response to dopamine and acetylcholine co-stimulation in living flies. Experiments were carried out with both sexes, or with either sex. The data show that amnesiac is necessary for the PKA activation process that results from coincidence detection in the MB. Since PACAP peptide is cleaved by the human membrane neprilysin hNEP, an interaction was sought between Amnesiac and Neprilysin 1 (Nep1), a fly neprilysin involved in memory. When Nep1 expression is acutely knocked down in adult MB, memory deficits displayed by amn hypomorphic mutants are rescued. Consistently, Nep1 inhibition also restores normal PKA activation in amn mutant flies. Taken together the results suggest that Nep1 targets Amnesiac degradation in order to terminate its signaling function. This work thus highlights a key role for Amnesiac in establishing within the MB the PKA dynamics that sustain middle-term memory formation, a function modulated by Nep1.
Yang, S. Z. and Wildonger, J. (2020). Golgi Outposts Locally Regulate Microtubule Orientation in Neurons but Are Not Required for the Overall Polarity of the Dendritic Cytoskeleton. Genetics. PubMed ID: 32265236
Microtubule-organizing centers (MTOCs) often play a central role in organizing the cellular microtubule networks that underlie cell function. In neurons, microtubules in axons and dendrites have distinct polarities. Dendrite-specific Golgi outposts, in particular multi-compartment outposts, have emerged as regulators of acentrosomal microtubule growth, raising the question of whether outposts contribute to establishing or maintaining the overall polarity of the dendritic microtubule cytoskeleton. Using a combination of genetic approaches and live imaging in a Drosophila model, this study found that dendritic microtubule polarity is unaffected by eliminating known regulators of Golgi-dependent microtubule organization including the cis-Golgi matrix protein GM130, the fly AKAP450 ortholog pericentrin-like protein (plp), and centrosomin (cnn). This indicates that Golgi outposts are not essential for the formation or maintenance of a dendrite-specific cytoskeleton. However, the over-expression of GM130, which promotes the formation of ectopic multi-compartment units, is sufficient to alter dendritic microtubule polarity. Axonal microtubule polarity is similarly disrupted by the presence of ectopic multi-compartment Golgi outposts. Notably, multi-compartment outposts alter microtubule polarity independently of microtubule nucleation mediated by the gamma-tubulin ring complex (gamma-TuRC). Thus, although Golgi outposts are not essential to dendritic microtubule polarity, altering their organization correlates with changes to microtubule polarity. Based on these data, it is proposed that the organization of Golgi outposts is carefully regulated to ensure proper dendritic microtubule polarity.
Wolterhoff, N., Gigengack, U. and Rumpf, S. (2020). PP2A phosphatase is required for dendrite pruning via actin regulation in Drosophila. EMBO Rep: e48870. PubMed ID: 32207238
Large-scale pruning, the developmentally regulated degeneration of axons or dendrites, is an important specificity mechanism during neuronal circuit formation. The peripheral sensory class IV dendritic arborization (c4da) neurons of Drosophila larvae specifically prune their dendrites at the onset of metamorphosis in an ecdysone-dependent manner. Dendrite pruning requires local cytoskeleton remodeling, and the actin-severing enzyme Mical is an important ecdysone target. In a screen for pruning factors, this study identified the protein phosphatase 2 A (PP2A). PP2A interacts genetically with the actin-severing enzymes Mical and cofilin as well as other actin regulators during pruning. Moreover, Drosophila cofilin undergoes a change in localization at the onset of metamorphosis indicative of a change in actin dynamics. This change is abolished both upon loss of Mical and PP2A. It is concluded that PP2A regulates actin dynamics during dendrite pruning.
Ueno, K., Morstein, J., Ofusa, K., Naganos, S., Suzuki-Sawano, E., Minegishi, S., Rezgui, S. P., Kitagishi, H., Michel, B. W., Chang, C. J., Horiuchi, J. and Saitoe, M. (2020). Carbon Monoxide, a Retrograde Messenger Generated in Postsynaptic Mushroom Body Neurons, Evokes Noncanonical Dopamine Release. J Neurosci 40(18): 3533-3548. PubMed ID: 32253360
Dopaminergic neurons innervate extensive areas of the brain and release dopamine (DA) onto a wide range of target neurons. However, DA release is also precisely regulated. In Drosophila melanogaster brain explant preparations, DA is released specifically onto alpha3/alpha'3 compartments of mushroom body (MB) neurons that have been coincidentally activated by cholinergic and glutamatergic inputs. The mechanism for this precise release has been unclear. This study found that coincidentally activated MB neurons generate carbon monoxide (CO), which functions as a retrograde signal evoking local DA release from presynaptic terminals. CO production depends on activity of heme oxygenase in postsynaptic MB neurons, and CO-evoked DA release requires Ca(2+) efflux through ryanodine receptors in DA terminals. CO is only produced in MB areas receiving coincident activation, and removal of CO using scavengers blocks DA release. It is proposed that DA neurons use two distinct modes of transmission to produce global and local DA signaling.
Alpert, M. H., Frank, D. D., Kaspi, E., Flourakis, M., Zaharieva, E. E., Allada, R., Para, A. and Gallio, M. (2020). A Circuit Encoding Absolute Cold Temperature in Drosophila. Curr Biol. PubMed ID: 32442464
Animals react to environmental changes over timescales ranging from seconds to days and weeks. An important question is how sensory stimuli are parsed into neural signals operating over such diverse temporal scales. This study uncover a specialized circuit, from sensory neurons to higher brain centers, that processes information about long-lasting, absolute cold temperature in Drosophila. Second-order thermosensory projection neurons (TPN-IIs) were identified exhibiting sustained firing that scales with absolute temperature. Strikingly, this activity only appears below the species-specific, preferred temperature for D. melanogaster (∼25°C). The inputs and outputs of TPN-IIs were traced, and they were found to be embedded in a cold "thermometer" circuit that provides powerful and persistent inhibition to brain centers involved in regulating sleep and activity. These results demonstrate that the fly nervous system selectively encodes and relays absolute temperature information and illustrate a sensory mechanism that allows animals to adapt behavior specifically to cold conditions on the timescale of hours to days.
Azevedo, A. W., Dickinson, E. S., Gurung, P., Venkatasubramanian, L., Mann, R. S. and Tuthill, J. C. (2020). A size principle for recruitment of Drosophila leg motor neurons. Elife 9. PubMed ID: 32490810

To move the body, the brain must precisely coordinate patterns of activity among diverse populations of motor neurons. This study used in vivo calcium imaging, electrophysiology, and behavior to understand how genetically-identified motor neurons control flexion of the fruit fly tibia. Leg motor neurons exhibit a coordinated gradient of anatomical, physiological, and functional properties. Large, fast motor neurons control high force, ballistic movements while small, slow motor neurons control low force, postural movements. Intermediate neurons fall between these two extremes. This hierarchical organization resembles the size principle, first proposed as a mechanism for establishing recruitment order among vertebrate motor neurons. Recordings in behaving flies confirmed that motor neurons are typically recruited in order from slow to fast. However, this study also found that fast, intermediate, and slow motor neurons receive distinct proprioceptive feedback signals, suggesting that the size principle is not the only mechanism that dictates motor neuron recruitment. Overall, this work reveals the functional organization of the fly leg motor system and establishes Drosophila as a tractable system for investigating neural mechanisms of limb motor control.

Monday, June 15th - Signaling

Ngo, S., Liang, J., Su, Y. H. and O'Brien, L. E. (2020). Disruption of EGF Feedback by Intestinal Tumors and Neighboring Cells in Drosophila. Curr Biol 30(8): 1537-1546. PubMed ID: 32243854
In healthy adult organs, robust feedback mechanisms control cell turnover to enforce homeostatic equilibrium between cell division and death. Nascent tumors must subvert these mechanisms to achieve cancerous overgrowth. Elucidating the nature of this subversion can reveal how cancers become established and may suggest strategies to prevent tumor progression. In adult Drosophila intestine, a well-studied model of homeostatic cell turnover, the linchpin of cell equilibrium is feedback control of the epidermal growth factor (EGF) protease Rhomboid (Rho). Expression of Rho in apoptotic cells enables them to secrete EGFs, which stimulate nearby stem cells to undergo replacement divisions. As in mammals, loss of adenomatous polyposis coli (APC) causes Drosophila intestinal stem cells to form adenomas. This study demonstrates that Drosophila APC(-/-) tumors trigger widespread Rho expression in non-apoptotic cells, resulting in chronic EGF signaling. Initially, nascent APC(-/-) tumors induce rho in neighboring wild-type cells via acute, non-autonomous activation of Jun N-terminal kinase (JNK). During later growth and multilayering, APC(-/-) tumors induce rho in tumor cells by autonomous downregulation of E-cadherin (E-cad) and consequent activity of p120-catenin. This sequential dysregulation of tumor non-autonomous and -autonomous EGF signaling converts tissue-level feedback into feed-forward activation that drives cancerous overgrowth. Because Rho, EGF receptor (EGFR), and E-cad are associated with colorectal cancer in humans, these findings may shed light on how human colorectal tumors progress.
Yuan, D., Zhou, S., Liu, S., Li, K., Zhao, H., Long, S., Liu, H., Xie, Y., Su, Y., Yu, F. and Li, S. (2020). The AMPK-PP2A axis in insect fat body is activated by 20-hydroxyecdysone to antagonize insulin/IGF signaling and restrict growth rate. Proc Natl Acad Sci U S A 117(17): 9292-9301. PubMed ID: 32277029
In insects, 20-hydroxyecdysone (20E) limits the growth period by triggering developmental transitions; 20E also modulates the growth rate by antagonizing insulin/insulin-like growth factor signaling (IIS). Previous work has shown that 20E cross-talks with IIS, but the underlying molecular mechanisms are not fully understood. This study found that, in both the silkworm Bombyx mori and the fruit fly Drosophila melanogaster, 20E antagonized IIS through the AMP-activated protein kinase (AMPK)-protein phosphatase 2A (PP2A) axis in the fat body and suppressed the growth rate. During Bombyx larval molt or Drosophila pupariation, high levels were found of 20E activate AMPK, a molecular sensor that maintains energy homeostasis in the insect fat body. In turn, AMPK activates PP2A, which further dephosphorylates insulin receptor and protein kinase B (AKT), thus inhibiting IIS. Activation of the AMPK-PP2A axis and inhibition of IIS in the Drosophila fat body reduced food consumption, resulting in the restriction of growth rate and body weight. Overall, this study revealed an important mechanism by which 20E antagonizes IIS in the insect fat body to restrict the larval growth rate, thereby expanding understanding of the comprehensive regulatory mechanisms of final body size in animals.
Woodling, N. S., Aleyakpo, B., Dyson, M. C., Minkley, L. J., Rajasingam, A., Dobson, A. J., Leung, K. H. C., Pomposova, S., Fuentealba, M., Alic, N. and Partridge, L. (2020). The neuronal receptor tyrosine kinase Alk is a target for longevity. Aging Cell: e13137. PubMed ID: 32291952
Inhibition of signalling through several receptor tyrosine kinases (RTKs), including the insulin-like growth factor receptor and its orthologues, extends healthy lifespan in organisms from diverse evolutionary taxa. This raises the possibility that other RTKs, including those already well studied for their roles in cancer and developmental biology, could be promising targets for extending healthy lifespan. This study focused on anaplastic lymphoma kinase (Alk), an RTK with established roles in nervous system development and in multiple cancers, but whose effects on aging remain unclear. Several means of reducing Alk signalling, including mutation of its ligand jelly belly (jeb), RNAi knock-down of Alk, or expression of dominant-negative Alk in adult neurons, can extend healthy lifespan in female, but not male, Drosophila. Moreover, reduced Alk signalling preserves neuromuscular function with age, promotes resistance to starvation and xenobiotic stress, and improves night sleep consolidation. Further it was found that inhibition of Alk signalling in adult neurons modulates the expression of several insulin-like peptides, providing a potential mechanistic link between neuronal Alk signalling and organism-wide insulin-like signalling. Finally, TAE-684, a small molecule inhibitor of Alk, can extend healthy lifespan in Drosophila, suggesting that the repurposing of Alk inhibitors may be a promising direction for strategies to promote healthy aging.
Syal, S., Ng, C., Kim, Y., Janbieh, J., Govind, S. and Deshpande, G. (2020). Reactive oxygen species signaling in primordial germ cell development in Drosophila embryos. Genesis: e23362. PubMed ID: 32302036
REDOX mechanisms that induce biosynthesis of the reactive oxygen species (ROS) have attracted considerable attention due to both the deleterious and beneficial responses elicited by the reactive radical. In several organisms including Drosophila melanogaster, modulation of ROS activity is thought to be crucial for the maintenance of cell fates in developmental contexts. Interestingly, REDOX mechanisms have been shown to be involved in maintaining progenitor fate of stem cells as well as their proliferation and differentiation. This study has explored the possible functions of ROS during proper specification and developmental progression of embryonic primordial germ cells (PGCs). Indicating its potential involvement in these processes, ROS can be detected in the embryonic PGCs and the surrounding somatic cells from very early stages of embryogenesis. Using both "loss" and "gain" of function mutations in two different components of the REDOX pathway, this study shows that ROS levels are likely to be critical in maintaining germ cell behavior, including their directed migration. Altering the activity of a putative regulator of ROS also adversely influences the ability of PGCs to adhere to one another in cellular blastoderm embryos, suggesting potential involvement of this pathway in orchestrating different phases of germ cell migration.
Zhou, J. and Boutros, M. (2020). JNK-dependent intestinal barrier failure disrupts host-microbe homeostasis during tumorigenesis. Proc Natl Acad Sci U S A 117(17): 9401-9412. PubMed ID: 32277031
In all animals, the intestinal epithelium forms a tight barrier to the environment. The epithelium regulates the absorption of nutrients, mounts immune responses, and prevents systemic infections. This study investigate the consequences of tumorigenesis on the microbiome using a Drosophila intestinal tumor model. Upon loss of BMP signaling, tumors lead to aberrant activation of JNK/Mmp2 signaling, followed by intestinal barrier dysfunction and commensal imbalance. In turn, the dysbiotic microbiome triggers a regenerative response and stimulates tumor growth. Inhibiting JNK signaling or depletion of the microbiome restores barrier function of the intestinal epithelium, leading to a reestablishment of host-microbe homeostasis, and organismic lifespan extension. These experiments identify a JNK-dependent feedback amplification loop between intestinal tumors and the microbiome. They also highlight the importance of controlling the activity level of JNK signaling to maintain epithelial barrier function and host-microbe homeostasis.
Wang, Q. P., Lin, Y. Q., Lai, M. L., Su, Z., Oyston, L. J., Clark, T., Park, S. J., Khuong, T. M., Lau, M. T., Shenton, V., Shi, Y. C., James, D. E., Ja, W. W., Herzog, H., Simpson, S. J. and Neely, G. G. (2020). PGC1alpha Controls Sucrose Taste Sensitization in Drosophila. Cell Rep 31(1): 107480. PubMed ID: 32268099
Perceived palatability of food controls caloric intake. Sweet taste is the primary means of detecting the carbohydrate content of food. Surprisingly, sweet taste sensitivity is responsive to extrinsic factors like diet, and this occurs by unknown mechanisms. This study describes an unbiased proteomic investigation into sweet taste sensitivity in the fruit fly. A dopamine/cyclic AMP (cAMP)/CREB axis acting within sweet taste neurons that controls taste perception but is largely dispensable for acute taste transduction. This pathway modulates sweet taste perception in response to both sensory- and nutrient-restricted diets and converges on PGC1alpha, a critical regulator of metabolic health and lifespan. By electrophysiology, it was found that enhanced sucrose taste sensitivity was the result of heightened sweet taste intensity and that PGC1alpha was both necessary and sufficient for this effect. Together, this study provides the first molecular insight into how diet-induced taste perception is regulated within the sweet taste neuron.

Friday June 12th - Evolution

Abhilash, L., Kalliyil, A. and Sheeba, V. (2020). Responses of activity rhythms to temperature cues evolve in Drosophila populations selected for divergent timing of eclosion. J Exp Biol. PubMed ID: 32291322
Even though the rhythm in adult emergence and rhythm in locomotor activity are two different rhythmic phenomena that occur at distinct life-stages of the fly life cycle, previous studies have hinted at similarities in certain aspects of the organisation of the circadian clock driving these two rhythms. For instance, the period gene plays an important regulatory role in both rhythms. Previous work showed that selection on timing of adult emergence behaviour in populations of Drosophila melanogaster leads to the co-evolution of temperature sensitivity of circadian clocks driving eclosion. In this study, it was asked if temperature sensitivity of the locomotor activity rhythm has evolved in the selected populations with divergent timing of adult emergence rhythm, with the goal of understanding the extent of similarity (or lack of it) in circadian organisation between the two rhythms. In response to simulated jetlag with temperature cycles, late chronotypes (populations selected for predominant emergence during dusk) indeed re-entrain faster than early chronotypes (populations selected for predominant emergence during dawn) to 6-h phase-delays, thereby indicating enhanced sensitivity of the activity/rest clock to temperature cues in these stocks (entrainment is the synchronisation of internal rhythms to cyclic environmental time-cues). Additionally, it was found that late chronotypes show higher plasticity of phases across regimes, day-to-day stability in phases and amplitude of entrainment, all indicative of enhanced temperature sensitive activity/rest rhythms. These results highlight remarkably similar organisation principles between emergence and activity/rest rhythms.
Xu, L. C., Nunes, C., Wang, V. R., Saito, A., Chen, T., Basak, P., Chang, J. J., Koyama, T. and Suzuki, Y. (2020). Distinct nutritional and endocrine regulation of prothoracic gland activities underlies divergent life history strategies in Manduca sexta and Drosophila melanogaster. Insect Biochem Mol Biol 119: 103335. PubMed ID: 32061770
Life history trade-offs lead to various strategies that maximize fitness, but the developmental mechanisms underlying these alternative strategies continue to be poorly understood. In insects, trade-offs exist between size and developmental time. Recent studies in the fruit fly Drosophila melanogaster have suggested that the steroidogenic prothoracic glands play a key role in determining the timing of metamorphosis. In this study, the nutrient-dependent growth and transcriptional activation of prothoracic glands were studied in D. melanogaster and the tobacco hornworm Manduca sexta. In both species, minimum viable weight (MVW) was associated with activation of ecdysteroid biosynthesis genes and growth of prothoracic gland cells. However, the timing of MVW attainment in M. sexta is delayed by the presence of the sesquiterpenoid hormone, juvenile hormone (JH), whereas in D. melanogaster it is not. Moreover, in D. melanogaster, the transcriptional regulation of ecdysteroidogenesis becomes nutrient-independent at the MVW/critical weight (CW) checkpoint. In contrast, in M. sexta, starvation consistently reduced transcriptional activation of ecdysteroid biosynthesis genes even after CW attainment, indicating that the nature of CW differs fundamentally between the two species. In D. melanogaster, the prothoracic glands dictate the timing of metamorphosis even in the absence of nutritional inputs, whereas in M. sexta, prothoracic gland activity is tightly coupled to the nutritional status of the body, thereby delaying the onset of metamorphosis before CW attainment. It is proposed that selection for survival under unpredictable nutritional availability leads to the evolution of increased modularity in both morphological and endocrine traits.
Oberhofer, G., Ivy, T. and Hay, B. A. (2020). Gene drive and resilience through renewal with next generation Cleave and Rescue selfish genetic elements. Proc Natl Acad Sci U S A 117(16): 9013-9021. PubMed ID: 32245808
Gene drive-based strategies for modifying populations face the problem that genes encoding cargo and the drive mechanism are subject to separation, mutational inactivation, and loss of efficacy. Resilience, an ability to respond to these eventualities in ways that restore population modification with functional genes, is needed for long-term success. This study shows that resilience can be achieved through cycles of population modification with "Cleave and Rescue" (ClvR) selfish genetic elements. ClvR comprises a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene and a recoded version of the essential gene resistant to cleavage. ClvR spreads by creating conditions in which those lacking ClvR die because they lack functional versions of the essential gene. Cycles of modification can, in principle, be carried out if two ClvR elements targeting different essential genes are located at the same genomic position, and one of them, ClvR (n+1), carries a Rescue transgene from an earlier element, ClvR (n) ClvR (n+1) should spread within a population of ClvR (n), while also bringing about a decrease in its frequency. To test this hypothesis, it was first shown that multiple ClvRs, each targeting a different essential gene, function when located at a common chromosomal position in Drosophila. It was then shown that when several of these also carry the Rescue from a different ClvR, they spread to transgene fixation in populations fixed for the latter and at its expense. Therefore, genetic modifications of populations can be overwritten with new content, providing an ongoing point of control.
Vorster, P. J., Goetsch, P., Wijeratne, T. U., Guiley, K. Z., Andrejka, L., Tripathi, S., Larson, B. J., Rubin, S. M., Strome, S. and Lipsick, J. S. (2020). A long lost key opens an ancient lock: Drosophila Myb causes a synthetic multivulval phenotype in nematodes. Biol Open. PubMed ID: 32295830
The five-protein MuvB core complex is highly conserved in animals. This nuclear complex interacts with RB family tumor suppressor proteins and E2F-DP transcription factors to form DREAM complexes that repress genes that regulate cell cycle progression and cell fate. The MuvB core complex also interacts with proteins Myb family oncoproteins to form the Myb-MuvB complexes that activate many of the same genes. This study shows that animal-type Myb genes are present in Bilateria, Cnidaria, and Placozoa, the latter including the simplest known animal species. However, bilaterian nematode worms lost their animal-type Myb genes hundreds of millions of years ago. Nevertheless, amino acids in the LIN9 and LIN52 proteins that directly interact with the MuvB-binding domains of human B-Myb and Drosophila Myb are conserved in C. elegans. Despite greater than 500 million years since their last common ancestor, the Drosophila melanogaster Myb protein can bind to the nematode LIN9-LIN52 proteins in vitro and can cause a synthetic multivulval (synMuv) phenotype in vivo. This phenotype is similar to that caused by loss-of-function mutations in C. elegans synMuvB class genes including those that encode homologs of the MuvB core, RB, E2F, and DP. Furthermore, amino acid substitutions in the MuvB-binding domain of Drosophila Myb that disrupt its functions in vitro and in vivo also disrupt these activities in C. elegans We speculate that nematodes and other animals may contain another protein that can bind to LIN9 and LIN52 in order to activate transcription of genes repressed by DREAM complexes.
Sergeeva, A. P., Katsamba, P. S., Cosmanescu, F., Brewer, J. J., Ahlsen, G., Mannepalli, S., Shapiro, L. and Honig, B. (2020). DIP/Dpr interactions and the evolutionary design of specificity in protein families.. Nat Commun 11(1): 2125. PubMed ID: 32358559
Differential binding affinities among closely related protein family members underlie many biological phenomena, including cell-cell recognition. Drosophila DIP and Dpr proteins mediate neuronal targeting in the fly through highly specific protein-protein interactions. This study shows that DIPs/Dprs segregate into seven specificity subgroups defined by binding preferences between their DIP and Dpr members. Then, a sequence-, structure- and energy-based computational approach, combined with experimental binding affinity measurements, is described to reveal how specificity is coded on the canonical DIP/Dpr interface. Binding specificity of DIP/Dpr subgroups is controlled by "negative constraints", which interfere with binding. To achieve specificity, each subgroup utilizes a different combination of negative constraints, which are broadly distributed and cover the majority of the protein-protein interface. The structural origins are described of negative constraints, and potential general implications for the evolutionary origins of binding specificity in multi-protein families.
Heames, B., Schmitz, J. and Bornberg-Bauer, E. (2020). A Continuum of Evolving De Novo Genes Drives Protein-Coding Novelty in Drosophila. J Mol Evol 88(4): 382-398. PubMed ID: 32253450
Orphan genes, lacking detectable homologs in outgroup species, typically represent 10-30% of eukaryotic genomes. This study investigated the roots of orphan gene emergence in the Drosophila genus. Across the annotated proteomes of twelve species, 6297 orphan genes were found within 4953 taxon-specific clusters of orthologs. By inferring the ancestral DNA as non-coding for between 550 and 2467 (8.7-39.2%) of these genes, this study describes for the first time how de novo emergence contributes to the abundance of clade-specific Drosophila genes. In support of them having functional roles, it was shown that de novo genes have robust expression and translational support. However, the distinct nucleotide sequences of de novo genes, which have characteristics intermediate between intergenic regions and conserved genes, reflect their recent birth from non-coding DNA. It was found that de novo genes encode more disordered proteins than both older genes and intergenic regions. Together, these results suggest that gene emergence from non-coding DNA provides an abundant source of material for the evolution of new proteins. Following gene birth, gradual evolution over large evolutionary timescales moulds sequence properties towards those of conserved genes, resulting in a continuum of properties whose starting points depend on the nucleotide sequences of an initial pool of novel genes.

Thursday June 11th, Adult Development

Zhang, R., Zhang, Z., Huang, Y., Qian, A. and Tan, A. (2020). A single ortholog of teashirt and tiptop regulates larval pigmentation and adult appendage patterning in Bombyx mori. Insect Biochem Mol Biol 121: 103369. PubMed ID: 32243904
Two paralogous genes, teashirt (tsh) and tiptop (tio), encode zinc-finger transcription factors and play important roles in insect growth and development. In the fruit fly, Drosophila melanogaster, tsh promotes trunk segmental identities and contributes to the patterning of other tissues during the embryonic stage. During the adult stage, tsh contributes to the specification and patterning of appendages, including the leg, wing and eye. While tio acts redundantly with tsh, flies lacking tio function are viable without deleterious phenotypes. This gene pair is present in the genomes of all Drosophila species but only as a single homologue in several other insect species. In Oncopeltus fasciatus and Tribolium castaneum, tsh/tio has been functionally characterized as specifying the identity of the leg during the adult stage. However, in lepidopteran insects which include large numbers of pests in agriculture and forestry, as well as the major silk producer silkworm Bombyx mori, the biological functions of tsh/tio are still poorly understood. This study performed functional analysis of tsh/tio by using both CRISPR/Cas9-mediated mutagenesis and transposon-mediated ectopic expression in B. mori. The results show that loss of tsh/tio function affected pigmentation during the larval stage and appendage pattering during the adult stage. RNA-seq analysis and subsequent q-RT-PCR analysis revealed that depletion of tsh/tio significantly elevated the expression of the kynurenine 3-monooxygenase gene, as well as melanin synthase-related genes during the larval stage. Furthermore, ubiquitous ectopic expression of tsh/tio induces developmental retardation and eventually larval lethality. These data reveal evolutionarily conserved functions of tsh/tio in controlling adult appendage patterning, as well as the novel function of regulating larval pigmentation in B. mori, providing novel insights into how tsh/tio regulates insect growth and development.
Zeng, J., Huynh, N., Phelps, B. and King-Jones, K. (2020). Snail synchronizes endocycling in a TOR-dependent manner to coordinate entry and escape from endoreplication pausing during the Drosophila critical weight checkpoint. PLoS Biol 18(2): e3000609. PubMed ID: 32097403
In holometabolous insects, the growth period is terminated through a cascade of peptide and steroid hormones that end larval feeding behavior and trigger metamorphosis, a nonfeeding stage during which the larval body plan is remodeled to produce an adult. This irreversible decision, termed the critical weight (CW) checkpoint, ensures that larvae have acquired sufficient nutrients to complete and survive development to adulthood. How insects assess body size via the CW checkpoint is still poorly understood on the molecular level. This study shows that the Drosophila transcription factor Snail plays a key role in this process. Before and during the CW checkpoint, snail is highly expressed in the larval prothoracic gland (PG), an endocrine tissue undergoing endoreplication and primarily dedicated to the production of the steroid hormone ecdysone. Two Snail peaks were observed in the PG, one before and one after the molt from the second to the third instar. Remarkably, these Snail peaks coincide with two peaks of PG cells entering S phase and a slowing of DNA synthesis between the peaks. Interestingly, the second Snail peak occurs at the exit of the CW checkpoint. Snail levels then decline continuously, and endoreplication becomes nonsynchronized in the PG after the CW checkpoint. This suggests that the synchronization of PG cells into S phase via Snail represents the mechanistic link used to terminate the CW checkpoint. Indeed, PG-specific loss of snail function prior to the CW checkpoint causes larval arrest due to a cessation of endoreplication in PG cells, whereas impairing snail after the CW checkpoint no longer affected endoreplication and further development. During the CW window, starvation or loss of TOR signaling disrupted the formation of Snail peaks and endocycle synchronization, whereas later starvation had no effect on snail expression. Taken together, these data demonstrate that insects use the TOR pathway to assess nutrient status during larval development to regulate Snail in ecdysone-producing cells as an effector protein to coordinate endoreplication and CW attainment.
Wang, S. J. H., Sinclair, D. A. R., Kim, H. Y., Kinsey, S. D., Yoo, B., Shih, C. R. Y., Wong, K. K. L., Krieger, C., Harden, N. and Verheyen, E. M. (2020). Homeodomain-interacting protein kinase (Hipk) plays roles in nervous system and muscle structure and function. PLoS One 15(3): e0221006. PubMed ID: 32187190
Homeodomain-interacting protein kinases (Hipks) have been previously associated with cell proliferation and cancer, however, their effects in the nervous system are less well understood. This study used Drosophila melanogaster to evaluate the effects of altered Hipk expression on the nervous system and muscle. Using genetic manipulation of Hipk expression it was demonstrated that knockdown and over-expression of Hipk produces early adult lethality, possibly due to the effects on the nervous system and muscle involvement. Optimal levels of Hipk are critical for the function of dopaminergic neurons and glial cells in the nervous system, as well as muscle. Furthermore, manipulation of Hipk affects the structure of the larval neuromuscular junction (NMJ) by promoting its growth. Hipk regulates the phosphorylation of the synapse-associated cytoskeletal protein Hu-li tai shao (Hts; adducin in mammals) and modulates the expression of two important protein kinases, Calcium-calmodulin protein kinase II (CaMKII) and Partitioning-defective 1 (PAR-1), all of which may alter neuromuscular structure/function and influence lethality. Hipk also modifies the levels of an important nuclear protein, TBPH, the fly orthologue of TAR DNA-binding protein 43 (TDP-43), which may have relevance for understanding motor neuron diseases.
Hughes, C. J. R., Turner, S., Andrews, R. M., Vitkin, A. and Jacobs, J. R. (2020). Matrix metalloproteinases regulate ECM accumulation but not larval heart growth in Drosophila melanogaster. J Mol Cell Cardiol 140: 42-55. PubMed ID: 32105665
If major cardiac extracellular matrix (ECM) components are secreted remotely, how is ECM "self assembly" regulated? This study explored whether ECM proteases were required to maintain the morphology of a growing heart while the cardiac ECM expanded. An increase in expression of Drosophila's single tissue inhibitor of metalloproteinase (TIMP), or reduced function of metalloproteinase MMP2, resulted in fibrosis and ectopic deposition of two ECM Collagens; type-IV and fibrillar Pericardin. Significant accumulations of Collagen-IV (Viking) developed on the pericardium and in the lumen of the heart. Congenital defects in Pericardin deposition misdirected further assembly in the larva. Reduced metalloproteinase activity during growth also increased Pericardin fibre accumulation in ECM suspending the heart. Although MMP2 expression was required to remodel and position cardiomyocyte cell junctions, reduced MMP function did not impair expansion of the heart. A previous study revealed that MMP2 negatively regulates the size of the luminal cell surface in the embryonic heart. Cardiomyocytes align at the midline, but do not adhere to enclose a heart lumen in MMP2 mutant embryos. Nevertheless, these embryos hatch and produce viable larvae with bifurcated hearts, indicating a secondary pathway to lumen formation between ipsilateral cardiomyocytes. MMP-mediated remodelling of the ECM is required for organogenesis, and to prevent assembly of excess or ectopic ECM protein during growth. MMPs are not essential for normal growth of the Drosophila heart.
Gershman, B. W., Pritchard, C. E., Chaney, K. P. and Ware, V. C. (2020). Tissue-specific Expression of Ribosomal Protein Paralogue eRpL22-like in Drosophila melanogaster Eye Development. Dev Dyn. PubMed ID: 32353187
Differences in core or tissue-specific ribosomal protein (Rp) composition within ribosomes contribute to ribosome heterogeneity and functional variability. Yet, the degree to which ribosome heterogeneity modulates development is unknown. The Drosophila melanogaster eRpL22 family contains structurally diverse paralogues, eRpL22 and eRpL22-like. Unlike ubiquitously expressed eRpL22, eRpL22-like expression is tissue-specific, notably within the male germline and the eye. This study investigated expression within the developing eye to uncover tissue/cell types where specific paralogue roles might be defined. Immunohistochemistry analysis confirms ubiquitous eRpL22 expression throughout eye development. In larvae, eRpL22-like is ubiquitously expressed, but highly enriched in the peripodial epithelium (PE). In early pupae, eRpL22-like is broadly distributed in multiple cell types, but later, is primarily enriched in interommatidial hair cells (IoHC). Adult patterns include the ring of accessory cells around ommatidia. Adult retinae IoHC patterning phenotypes (shown by scanning electron microscopy) may be linked to RNAi-mediated eRpL22-like depletion within larval PE. Immunoblots and polysome profile analyses show multiple variants of eRpL22-like across development, with the variant at the expected molecular mass co-sedimenting with active ribosomes. These data reveal differential patterns of eRpL22-like expression relative to eRpL22 and suggest a specific role for eRpL22-like in developmental patterning of the eye.
Redhai, S., Pilgrim, C., Gaspar, P., Giesen, L. V., Lopes, T., Riabinina, O., Grenier, T., Milona, A., Chanana, B., Swadling, J. B., Wang, Y. F., Dahalan, F., Yuan, M., Wilsch-Brauninger, M., Lin, W. H., Dennison, N., Capriotti, P., Lawniczak, M. K. N., Baines, R. A., Warnecke, T., Windbichler, N., Leulier, F., Bellono, N. W. and Miguel-Aliaga, I. (2020). An intestinal zinc sensor regulates food intake and developmental growth. Nature 580(7802): 263-268. PubMed ID: 32269334
In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes. This study used a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes-interstitial cells-by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR-Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. These findings highlight the need to consider the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis.

Wednesday June 10th - Synapse and Vesicles

White, D., de Sousa Abreu, R. P., Blake, A., Murphy, J., Showell, S., Kitamoto, T. and Lawal, H. O. (2020). Deficits in the vesicular acetylcholine transporter alter lifespan and behavior in adult Drosophila melanogaster. Neurochem Int 137: 104744. PubMed ID: 32315665
The neurotransmitter acetylcholine (ACh) is involved in critical organismal functions that include locomotion and cognition. Importantly, alterations in the cholinergic system are a key underlying factor in cognitive defects associated with aging. One essential component of cholinergic synaptic transmission is the vesicular ACh transporter (VAChT), which regulates the packaging of ACh into synaptic vesicles for extracellular release. Mutations that cause a reduction in either protein level or activity lead to diminished locomotion ability whereas complete loss of function of VAChT is lethal. While much is known about the function of VAChT, the direct role of altered ACh release and its association with either an impairment or an enhancement of cognitive function are still not fully understood. It was hypothesized that point mutations in Vacht cause age-related deficits in cholinergic-mediated behaviors such as locomotion, and learning and memory. Using Drosophila melanogaster as a model system, several mutations within Vacht were studied and their effect on survivability and locomotive behavior were observed. A weak hypomorphic Vacht allele was found that shows a differential effect on ACh-linked behaviors. It was also demonstrated that partially rescued Vacht point mutations cause an allele-dependent deficit in lifespan and defects in locomotion ability. Moreover, using a thorough data analytics strategy to identify exploratory behavioral patterns, new paradigms were introduced for measuring locomotion-related activities that could not be revealed or detected by a simple measure of the average speed alone. Together, these data indicate a role for VAChT in the maintenance of longevity and locomotion abilities in Drosophila and additional measurements of locomotion are provided that can be useful in determining subtle changes in Vacht function on locomotion-related behaviors.
Wen, P., Zhang, F., Fu, Y., Zhu, J. Y. and Han, Z. (2020). Exocyst Genes Are Essential for Recycling Membrane Proteins and Maintaining Slit Diaphragm in Drosophila Nephrocytes. J Am Soc Nephrol 31(5): 1024-1034. PubMed ID: 32238475
Studies have linked mutations in genes encoding the eight-protein exocyst protein complex to kidney disease, but the underlying mechanism is unclear. Because Drosophila nephrocytes share molecular and structural features with mammalian podocytes, they provide an efficient model for studying this issue. Genes encoding exocyst complex proteins were silenced specifically in Drosophila nephrocytes, and the effects on protein reabsorption by lacuna channels and filtration by the slit diaphragm were studied. Nephrocyte functional assays were performed, super-resolution confocal microscopy of slit diaphragm proteins was carried out, and transmission electron microscopy was used to analyze ultrastructural changes. The colocalization of slit diaphragm proteins with exocyst protein Sec15 and with endocytosis and recycling regulators Rab5, Rab7, and Rab11 was also studied. Silencing exocyst genes in nephrocytes led to profound changes in structure and function. Abolition of cellular accumulation of hemolymph proteins with dramatically reduced lacuna channel membrane invaginations offered a strong indication of reabsorption defects. Moreover, the slit diaphragm's highly organized surface structure-essential for filtration-was disrupted, and key proteins were mislocalized. Ultrastructural analysis revealed that exocyst gene silencing led to the striking appearance of novel electron-dense structures that were named "exocyst rods," which likely represent accumulated membrane proteins following defective exocytosis or recycling. The slit diaphragm proteins partially colocalized with Sec15, Rab5, and Rab11. These findings suggest that the slit diaphragm of Drosophila nephrocytes requires balanced endocytosis and recycling to maintain its structural integrity and that impairment of the exocyst complex leads to disruption of the slit diaphragm and nephrocyte malfunction. This model may help identify therapeutic targets for treating kidney diseases featuring molecular defects in vesicle endocytosis, exocytosis, and recycling.
Perry, S., Goel, P., Tran, N. L., Pinales, C., Buser, C., Miller, D. L., Ganetzky, B. and Dickman, D. (2020). Developmental arrest of Drosophila larvae elicits presynaptic depression and enables prolonged studies of neurodegeneration. Development. PubMed ID: 32345746
Synapses exhibit an astonishing degree of adaptive plasticity in healthy and disease states. This study has investigated whether synapses also adjust to life stages imposed by novel developmental programs for which they were never molded by evolution. Under conditions where Drosophila larvae are terminally arrested, this study has characterized synaptic growth, structure and function at the neuromuscular junction (NMJ). While wild-type larvae transition to pupae after 5 days, arrested third instar (ATI) larvae persist for 35 days, during which NMJs exhibit extensive overgrowth in muscle size, presynaptic release sites, and postsynaptic glutamate receptors. Remarkably, despite this exuberant growth, stable neurotransmission is maintained throughout the ATI lifespan through a potent homeostatic reduction in presynaptic neurotransmitter release. Arrest of the larval stage in stathmin mutants also reveals a degree of progressive instability and neurodegeneration that was not apparent during the typical larval period. Hence, an adaptive form of presynaptic depression stabilizes neurotransmission during an extended developmental period of unconstrained synaptic growth. More generally, the ATI manipulation provides a powerful system for studying neurodegeneration and plasticity across prolonged developmental timescales.
Strah, N., Romano, G., Introna, C., Klima, R., Marzullo, M., Ciapponi, L., Megighian, A., Nizzardo, M. and Feiguin, F. (2020). TDP-43 promotes the formation of neuromuscular synapses through the regulation of Disc-large expression in Drosophila skeletal muscles. BMC Biol 18(1): 34. PubMed ID: 32216790
The ribonuclear protein TDP-43 has been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS), with genetic mutations being linked to the neurological symptoms of the disease. Alterations in the intracellular distribution of TDP-43 have been observed in skeletal muscles of patients suffering from ALS and the molecular and metabolic pathways regulated by TDP-43 in the skeletal muscle remain largely unknown. This study analyzed the function of TBPH, the Drosophila melanogaster ortholog of TDP-43, in skeletal muscles. The activity of TDP-43 in Drosophila muscles was modulated by means of RNA interference, and it was observed to be required to promote the formation and growth of neuromuscular synapses. TDP-43 regulated the expression levels of Disc-large (Dlg), and restoring Dlg expression either in skeletal muscles or in motoneurons was sufficient to suppress the locomotive and synaptic defects of TDP-43-null flies. These results were validated by the observation of a decrease in Dlg levels in human neuroblastoma cells and iPSC-differentiated motoneurons derived from ALS patients, suggesting similar mechanisms may potentially be involved in the pathophysiology of the disease. These results help to unveil the physiological role of TDP-43 in skeletal muscles as well as the mechanisms responsible for the autonomous and non-autonomous behavior of this protein concerning the organization of neuromuscular synapses.
Guan, Z., Quinones-Frias, M. C., Akbergenova, Y. and Littleton, J. T. (2020). Drosophila Synaptotagmin 7 negatively regulates synaptic vesicle release and replenishment in a dosage-dependent manner. Elife 9. PubMed ID: 32343229
Synchronous neurotransmitter release is triggered by Ca(2+) binding to the synaptic vesicle protein Synaptotagmin 1, while asynchronous fusion and short-term facilitation is hypothesized to be mediated by plasma membrane-localized Synaptotagmin 7 (SYT7). This study generated mutations in Drosophila Syt7 to determine if it plays a conserved role as the Ca(2+) sensor for these processes. Electrophysiology and quantal imaging revealed evoked release was elevated 2-fold. Syt7 mutants also had a larger pool of readily-releasable vesicles, faster recovery following stimulation, and intact facilitation. Syt1/Syt7 double mutants displayed more release than Syt1 mutants alone, indicating SYT7 does not mediate the residual asynchronous release remaining in the absence of SYT1. SYT7 localizes to an internal membrane tubular network within the peri-active zone, but does not enrich at active zones. These findings indicate the two Ca(2+) sensor model of SYT1 and SYT7 mediating all phases of neurotransmitter release and facilitation is not applicable at Drosophila synapses.
Odierna, G. L., Kerwin, S. K., Harris, L. E., Shin, G. J., Lavidis, N. A., Noakes, P. G. and Millard, S. S. (2020). Dscam2 suppresses synaptic strength through a PI3K-dependent endosomal pathway. J Cell Biol 219(6). PubMed ID: 32259198
Dscam2 is a cell surface protein required for neuronal development in Drosophila; it can promote neural wiring through homophilic recognition that leads to either adhesion or repulsion between neurites. This study reports that Dscam2 also plays a post-developmental role in suppressing synaptic strength. This function is dependent on one of two distinct extracellular isoforms of the protein and is autonomous to motor neurons. The PI3K enhancer, Centaurin gamma 1A, was linked to the Dscam2-dependent regulation of synaptic strength, and it was shown that changes in phosphoinositide levels correlate with changes in endosomal compartments that have previously been associated with synaptic strength. Using transmission electron microscopy, an increase was found in synaptic vesicles at Dscam2 mutant active zones, providing a rationale for the increase in synaptic strength. This study provides the first evidence that Dscam2 can regulate synaptic physiology and highlights how diverse roles of alternative protein isoforms can contribute to unique aspects of brain development and function.

Tuesday, June 9th - Behavior

Mathejczyk, T. F. and Wernet, M. F. (2020). Modular assays for the quantitative study of visually guided navigation in both flying and walking flies. J Neurosci Methods: 108747. PubMed ID: 32339523
The quantitative study of behavioral responses to visual stimuli provides crucial information about the computations executed by neural circuits. Insects have long served as powerful model systems, either when walking on air suspended balls (spherical treadmill), or flying while glued to a needle (virtual flight arena). This study presents detailed instructions for 3D-printing and assembly of arenas optimized for visually guided navigation, including codes for presenting both celestial and panorama cues. These modular arenas can be used either as virtual flight arenas, or as spherical treadmills and consist entirely of commercial and 3D-printed components placed in a temperature and humidity controlled environment. Robust optomotor responses are induced in flying Drosophila by displaying moving stripes in a cylinder surrounding the magnetically tethered fly. Similarly, changes in flight heading are induced by presenting changes in the orientation of linearly polarized UV light presented from above. Finally, responses to moving patterns are induced when individual flies are walking on an air-suspended ball. These modular assays allow for the investigation of a diverse combination navigational cues (sky and panorama) in both flying and walking flies. They can be used for the molecular dissection of neural circuitry in Drosophila and can easily be rescaled for accommodating other insects.
Schilling, M. and Cruse, H. (2020). Decentralized control of insect walking: A simple neural network explains a wide range of behavioral and neurophysiological results. PLoS Comput Biol 16(4): e1007804. PubMed ID: 32339162
Controlling the six legs of an insect walking in an unpredictable environment is a challenging task. Solutions proposed to deal with this task are usually based on the highly influential concept that (sensory-modulated) central pattern generators (CPG) are required to control the rhythmic movements of walking legs. This study investigated a different view. To this end, a sensor based controller operating on artificial neurons was introduced, being applied to a (simulated) insectoid robot required to exploit the 'loop through the world' allowing for simplification of neural computation. Such a decentralized solution is shown to lead to adaptive behavior when facing uncertain environments which are demonstrated for a broad range of behaviors never dealt with in a single system by earlier approaches. These patterns are found to be stable against disturbances and when starting from various leg configurations. This neuronal architecture easily allows for starting or interrupting a walk, all being difficult for CPG controlled solutions. This approach can as well account for the neurophysiological results usually interpreted to support the idea that CPGs form the basis of walking, although this approach is not relying on explicit CPG-like structures. Application of CPGs may however be required for very fast walking. This neuronal structure allows to pinpoint specific neurons known from various insect studies. Interestingly, specific common properties observed in both insects and crustaceans suggest a significance of the controller beyond the realm of insects
Showell, S. S., Martinez, Y., Gondolfo, S., Boppana, S. and Lawal, H. O. (2020). Overexpression of the vesicular acetylcholine transporter disrupts cognitive performance and causes age-dependent locomotion decline in Drosophila. Mol Cell Neurosci 105: 103483. PubMed ID: 32217162
Acetylcholinergic (ACh) neurotransmission is essential for key organismal functions such as locomotion and cognition. However, the mechanism through which ACh is regulated in the central nervous system is not fully understood. The vesicular acetylcholine transporter (VAChT) mediates the packaging and transport of ACh for exocytotic release and is a critical component of the ACh release machinery. Yet its precise role in the maintenance of cholinergic tone remains a subject of active investigation. This study use the overexpression of VAChT as a tool to investigate the role of changes in ACh exocytosis on the regulation of synaptic activity and its downstream consequences. Yhe effect was measured of an increase in VAChT expression on locomotion and cognitive performance as well as on organismal survival across the lifespan. The surprising finding is reported that increased VAChT expression results in a significantly shorter lifespan in comparison to control flies. Moreover, constructs overexpressing VAChT demonstrate an age-dependent decrease in locomotion performance. Importantly, this study reports clear deficits in learning and memory which wetr measured through a courtship conditioning assay. Together, these data provide evidence for the adverse effects of overexpression of the vesicular acetylcholine transporter in the maintenance of normal behavioral abilities in Drosophila and demonstrates for the first time a role for ACh in the regulation of organismal survival.
Tao, L., Ozarkar, S. and Bhandawat, V. (2020). Mechanisms underlying attraction to odors in walking Drosophila. PLoS Comput Biol 16(3): e1007718. PubMed ID: 32226007
Mechanisms that control movements range from navigational mechanisms, in which the animal employs directional cues to reach a specific destination, to search movements during which there are little or no environmental cues. Even though most real-world movements result from an interplay between these mechanisms, an experimental system and theoretical framework for the study of interplay of these mechanisms is not available. This study rectifies this deficit. A new method is created to stimulate the olfactory system in Drosophila. As flies explore a circular arena, their olfactory receptor neurons (ORNs) are optogenetically activated within a central region making this region attractive to the flies without emitting any clear directional signals outside this central region. In the absence of ORN activation, the fly's locomotion can be described by a random walk model where a fly's movement is described by its speed and turn-rate (or kinematics). Upon optogenetic stimulation, the fly's behavior changes dramatically in two respects. First, there are large kinematic changes. Second, there are more turns at the border between light-zone and no-light-zone and these turns have an inward bias. Surprisingly, there is no increase in turn-rate, rather a large decrease in speed that makes it appear that the flies are turning at the border. Similarly, the inward bias of the turns is a result of the increase in turn angle. These two mechanisms entirely account for the change in a fly's locomotion. No complex mechanisms such as path-integration or a careful evaluation of gradients are necessary.
Meda, N., Frighetto, G., Megighian, A. and Zordan, M. A. (2020). Searching for relief: Drosophila melanogaster navigation in a virtual bitter maze. Behav Brain Res: 112616. PubMed ID: 32361039
Animals use relief-based place learning to pinpoint a specific location where noxious stimuli are diminished or abolished. This study shows how the optogenetically-induced activation of bitter-sensing neurons in Drosophila melanogaster elicits pain-like behavioural responses and stimulates the search for a place where this activation is relieved. Under this 'virtual' stimulation paradigm it would be feasible to test relief learning several times throughout an animal's lifespan, without the potentially damaging effects which may result from the repeated administration of 'real' heat or electrical shock. Furthermore, virtual bitter taste could be used in place of virtual pain stimulation to guide conditioned place preference and study learning processes. It is also proposed that spatially-specific reduction of locomotor velocity may provide immediate evidence of relief-based place learning and spatial memory.
Shahandeh, M. P. and Turner, T. L. (2020). The complex genetic architecture of male mate choice evolution between Drosophila species. Heredity (Edinb). PubMed ID: 32203250
Mate choice behaviors are among the most important reproductive isolating barriers in many animals. Little is known about the genetic basis of reproductively isolating behaviors, but examples to date provide evidence that they can have a simple genetic basis. This study presents the results of a QTL mapping study for the most important behavioral isolating barrier between Drosophila simulans and D. sechellia: male mate choice. The QTL results initially suggested that differences in male mate choice may be due to a couple loci with large effects. However, as the largest-effect QTL was divided using stable introgression strains, evidence was found of multiple interacting loci. It was further found that separate regions of the genome control different aspects of male choice. Taken together, these results suggest that the genetic architecture of mate choice behavior, in this case, is more complex than QTL mapping suggested, highlighting potential challenges to future mapping studies. The implications of these results is discussed as they relate to signal-receiver coevolution, mate choice, and reproductive isolation.

Monday, June 8th - Adult neural development and function

Paglione, M., Rosell, A. L., Chatton, J. Y. and Neukomm, L. J. (2020). Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster. J Vis Exp(157). PubMed ID: 32225164
Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. This study presents three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. Examples are presented using a highwire loss-of-function mutation are presented, and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.
Rajagopalan, A. and Assisi, C. (2020). Effect of circuit structure on odor representation in the insect olfactory system. eNeuro. PubMed ID: 32345734
In Neuroscience, the structure of a circuit has often been used to intuit function - an inversion of Louis Kahn's famous dictum, 'Form follows function'. However, different brain networks may utilize different network architectures to solve the same problem. The olfactory circuits of two insects, the Locust, Schistocerca americana, and the fruit fly, Drosophila melanogaster, serve the same function - to identify and discriminate odors. The neural circuitry that achieves this shows marked structural differences. Projection neurons (PN) in the antennal lobe (AL) innervate Kenyon cells (KC) of the mushroom body (MB). In locust, each KC receives inputs from approximately 50% PNs, a scheme that maximizes the difference between inputs to any two of approximately 50,000 KCs. In contrast, in drosophila, this number is only 5% and appears sub-optimal. Using a computational model of the olfactory system, this study shows the activity of KCs is sufficiently high-dimensional that it can separate similar odors regardless of the divergence of PN-KC connections. However, when temporal patterning encodes odor attributes, dense connectivity outperforms sparse connections. Increased separability comes at the cost of reliability. The disadvantage of sparse connectivity can be mitigated by incorporating other aspects of circuit architecture seen in Drosophila. These simulations predict that Drosophila and locust circuits lie at different ends of a continuum where the Drosophila gives up on the ability to resolve similar odors to generalize across varying environments, while the locust separates odor representations but risks misclassifying noisy variants of the same odor.
Sabandal, J. M., Sabandal, P. R., Kim, Y. C. and Han, K. A. (2020). Concerted Actions of Octopamine and Dopamine Receptors Drive Olfactory Learning. J Neurosci. PubMed ID: 32277043
Aminergic signaling modulates associative learning and memory. Substantial advance has been made in Drosophila on the dopamine receptors and circuits mediating olfactory learning, however knowledge on other aminergic modulation lags behind. To address this knowledge gap, this study investigated the role of octopamine in olfactory conditioning. Octopamine activity through the beta adrenergic-like receptor Octbeta1R is shown to drive aversive and appetitive learning: Octbeta1R in the mushroom body alphabeta neurons processes aversive learning whereas Octbeta1R in the projection neurons mediates appetitive learning. Genetic interaction and imaging studies pinpoint cAMP signaling as a key downstream effector for Octbeta1R function. The rutabaga-adenylyl cyclase synthesizes cAMP in a Ca(2+)/calmodulin-dependent manner, serving as a coincidence detector for associative learning and likely representing a downstream target for Octbeta1R. Supporting this notion, the double heterozygous rutabaga/+;;octbeta1r/+ flies perform poorly in both aversive and appetitive conditioning, while individual heterozygous rutabaga/+ and octbeta1r/+ behave like the wild-type control. Consistently, the mushroom body and projection neurons in the octbeta1r brain exhibit blunted responses to octopamine when cAMP levels are monitored through the cAMP sensor. Previous work demonstrated the pivotal functions of the D1 receptor dDA1 in aversive and appetitive learning, and the alpha1 adrenergic-like receptor OAMB in appetitive learning. As expected, octbeta1r genetically interacts with dumb (dDA1 mutant) in aversive and appetitive learning, but it interacts with oamb only in appetitive learning. This study uncovers the indispensable contributions of dopamine and octopamine signaling to aversive and appetitive learning. All experiments were performed on mixed sex unless otherwise noted.
Salem, W., Cellini, B., Frye, M. A. and Mongeau, J. M. (2020). Fly eyes are not still: a motion illusion in Drosophila flight supports parallel visual processing. J Exp Biol. PubMed ID: 32321749
Most animals shift gaze by a 'fixate and saccade' strategy, where the fixation phase stabilizes background motion. A logical prerequisite for robust detection and tracking of moving foreground objects, therefore, is to suppress the perception of background motion. In a virtual reality magnetic tether system enabling free yaw movement, Drosophila implemented a fixate and saccade strategy in the presence of a static panorama. When the spatial wavelength of a vertical grating was below the Nyquist wavelength of the compound eyes, flies drifted continuously- and gaze could not be maintained at a single location. Because the drift occurs from a motionless stimulus-thus any perceived motion stimuli are generated by the fly itself-it is illusory, driven by perceptual aliasing. Notably, the drift speed was significantly faster than under a uniform panorama suggesting perceptual enhancement due to aliasing. Under the same visual conditions in a rigid tether paradigm, wing steering responses to the unresolvable static panorama were not distinguishable from a resolvable static pattern, suggesting visual aliasing is induced by ego motion. It is hypothesized that obstructing the control of gaze fixation also disrupts detection and tracking of objects. Using the illusory motion stimulus, it was shown that magnetically tethered Drosophila track objects robustly in flight even when gaze is not fixated as flies continuously drift. Taken together, this study provides further support for parallel visual motion processing and reveals the critical influence of body motion on visuomotor processing. Motion illusions can reveal important shared principles of information processing across taxa.
Shohayeb, B., Mitchell, N., Millard, S. S., Quinn, L. M. and Ng, D. C. H. (2020). Elevated levels of Drosophila Wdr62 promote glial cell growth and proliferation through AURKA signalling to AKT and MYC. Biochim Biophys Acta Mol Cell Res 1867(7): 118713. PubMed ID: 32246948
WD40-Repeat Protein 62 (WDR62) is required to maintain neural and glial cell populations during embryonic brain growth. Although elevated expression of WDR62 is frequently associated with several tumour types, potential effects of excess WDR62 on proliferative growth remain undefined. This study demonstrates that glia specific overexpression of WDR62 in Drosophila larval brains resulted in increased cell size, over-proliferation and increased brain volume, without overt disruption of tissue organization. It was further demonstrated that WDR62 promoted over-proliferation and brain overgrowth by activating AURKA and pAKT signalling to increase MYC function in glial cells. Together these data suggest WDR62 normally functions in the glial lineage to activate oncogenic signalling networks, promoting proliferation and brain overgrowth.
Li, Q., DeBeaubien, N. A., Sokabe, T. and Montell, C. (2020). Temperature and Sweet Taste Integration in Drosophila. Curr Biol. PubMed ID: 32330421
Sugar-containing foods offered at cooler temperatures tend to be less appealing to many animals. However, the mechanism through which the gustatory system senses thermal input and integrates temperature and chemical signals to produce a given behavioral output is poorly understood. To study this fundamental problem, the fly, Drosophila melanogaster, was used. It was found that the palatability of sucrose is strongly reduced by modest cooling. Using Ca(2+) imaging and electrophysiological recordings, it was demonstrated that bitter gustatory receptor neurons (GRNs) and mechanosensory neurons (MSNs) are activated by slight cooling, although sugar neurons are insensitive to the same mild stimulus. A rhodopsin, Rh6, is expressed and required in bitter GRNs for cool-induced suppression of sugar appeal. These findings reveal that the palatability of sugary food is reduced by slightly cool temperatures through different sets of thermally activated neurons, one of which depends on a rhodopsin (Rh6) for cool sensation.

Friday June 5th - Disease model

Kim, K., Lane, E. A., Saftien, A., Wang, H., Xu, Y., Wirtz-Peitz, F. and Perrimon, N. (2020). Drosophila as a model for studying cystic fibrosis pathophysiology of the gastrointestinal system. Proc Natl Acad Sci U S A. PubMed ID: 32345720
Cystic fibrosis (CF) is a recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The most common symptoms include progressive lung disease and chronic digestive conditions. CF is the first human genetic disease to benefit from having five different species of animal models. Despite the phenotypic differences among the animal models and human CF, these models have provided invaluable insight into understanding disease mechanisms at the organ-system level. This study identified a member of the ABCC4 family, CG5789, that has the structural and functional properties expected for encoding the Drosophila equivalent of human CFTR, and thus it is refered to as Drosophila CFTR (Dmel\CFTR). Knockdown of Dmel\CFTR in the adult intestine disrupts osmotic homeostasis and displays CF-like phenotypes that lead to intestinal stem cell hyperplasia. Expression of wild-type human CFTR, but not mutant variants of CFTR that prevent plasma membrane expression, rescues the mutant phenotypes of Dmel\CFTR. Furthermore, RNA sequencing (RNA-Seq)-based transcriptomic analysis was performed using Dmel\CFTR fly intestine and a mucin gene, Muc68D, was identified that is required for proper intestinal barrier protection. Altogether, these findings suggest that Drosophila can be a powerful model organism for studying CF pathophysiology.
Lim, C. H., Kaur, P., Teo, E., Lam, V. Y. M., Zhu, F., Kibat, C., Gruber, J., Mathuru, A. S. and Tolwinski, N. S. (2020). Application of optogenetic Amyloid-beta distinguishes between metabolic and physical damages in neurodegeneration. Elife 9. PubMed ID: 32228858
The brains of Alzheimer's disease patients show a decrease in brain mass and a preponderance of extracellular Amyloid-beta plaques. These plaques are formed by aggregation of polypeptides that are derived from the Amyloid Precursor Protein (APP). Amyloid-beta plaques are thought to play either a direct or an indirect role in disease progression, however the exact role of aggregation and plaque formation in the aetiology of Alzheimer's disease (AD) is subject to debate as the biological effects of soluble and aggregated Amyloid-beta peptides are difficult to separate in vivo. To investigate the consequences of formation of Amyloid-beta oligomers in living tissues, a fluorescently tagged, optogenetic Amyloid-beta peptide was developed that oligomerizes rapidly in the presence of blue light. This system was applied to the crucial question of how intracellular Amyloid-beta oligomers underlie the pathologies of A. Drosophila, C. elegans and D. rerio were used to show that, although both expression and induced oligomerization of Amyloid-beta were detrimental to lifespan and healthspan, it was possible to separate the metabolic and physical damage caused by light-induced Amyloid-beta oligomerization from Amyloid-beta expression alone. The physical damage caused by Amyloid-beta oligomers also recapitulated the catastrophic tissue loss that is a hallmark of late AD. The lifespan deficit induced by Amyloid-beta oligomers was reduced with Li(+) treatment. These results present the first model to separate different aspects of disease progression.
Kankel, M. W., Sen, A., Lu, L., Theodorou, M., Dimlich, D. N., McCampbell, A., Henderson, C. E., Shneider, N. A. and Artavanis-Tsakonas, S. (2020). Amyotrophic Lateral Sclerosis Modifiers in Drosophila Reveal the Phospholipase D Pathway as a Potential Therapeutic Target. Genetics. PubMed ID: 32345615
Amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease, is a devastating neurodegenerative disorder lacking effective treatments. ALS pathology is linked to mutations in more than twenty different genes indicating a complex underlying genetic architecture that is effectively unknown. In an attempt to identify genes and pathways for potential therapeutic intervention and explore the genetic circuitry underlying Drosophila models of ALS, this study carried out two independent genome-wide screens for modifiers of degenerative phenotypes associated with the expression of transgenic constructs carrying familial ALS (fALS)-causing alleles of FUS (hFUS(R521C)) and TDP-43 (hTDP-43(M337V)). A complex array of genes was uncovered affecting either - or both - of the two strains, and their activities were investigated in additional ALS models. These studies indicate the pathway that governs Phospholipase D (PLD) activity as a major modifier of ALS-related phenotypes, a notion supported by data generated in mice and humans.
Kanellopoulos, A. K., Mariano, V., Spinazzi, M., Woo, Y. J., McLean, C., Pech, U., Li, K. W., Armstrong, J. D., Giangrande, A., Callaerts, P., Smit, A. B., Abrahams, B. S., Fiala, A., Achsel, T. and Bagni, C. (2020). Aralar Sequesters GABA into Hyperactive Mitochondria, Causing Social Behavior Deficits. Cell 180(6): 1178-1197.e1120. PubMed ID: 32200800
Social impairment is frequently associated with mitochondrial dysfunction and altered neurotransmission. Although mitochondrial function is crucial for brain homeostasis, it remains unknown whether mitochondrial disruption contributes to social behavioral deficits. This study shows that Drosophila mutants in the homolog of the human CYFIP1, a gene linked to autism and schizophrenia, exhibit mitochondrial hyperactivity and altered group behavior. The regulation of GABA availability by mitochondrial activity was identified as a biologically relevant mechanism, and its contribution to social behavior was identified. Specifically, increased mitochondrial activity causes gamma aminobutyric acid (GABA) sequestration in the mitochondria, reducing GABAergic signaling and resulting in social deficits. Pharmacological and genetic manipulation of mitochondrial activity or GABA signaling corrects the observed abnormalities. Aralar was identified as the mitochondrial transporter that sequesters GABA upon increased mitochondrial activity. This study increases understanding of how mitochondria modulate neuronal homeostasis and social behavior under physiopathological conditions.
Petridi, S., Middleton, C. A., Ugbode, C., Fellgett, A., Covill, L. and Elliott, C. J. H. (2020). In Vivo Visual Screen for Dopaminergic Rab <--> LRRK2-G2019S Interactions in Drosophila Discriminates Rab10 from Rab3. G3 (Bethesda). PubMed ID: 32321836
LRRK2 mutations cause Parkinson's, but the molecular link from increased kinase activity to pathological neurodegeneration remains undetermined. Previous in vitro assays indicate that LRRK2 substrates include at least 8 Rab GTPases. This hypothesis was examined in vivo in a functional, electroretinogram screen, expressing each Rab with/without LRRK2-G2019S in selected Drosophila dopaminergic neurons. The screen discriminated Rab10 from Rab3. The strongest Rab/LRRK2-G2019S interaction is with Rab10; the weakest with Rab3. Rab10 is expressed in a different set of dopaminergic neurons from Rab3. Thus, anatomical and physiological patterns of Rab10 are related. It is concluded that Rab10 is a valid substrate of LRRK2 in dopaminergic neurons in vivo. It is proposed that variations in Rab expression contribute to differences in the rate of neurodegeneration recorded in different dopaminergic nuclei in Parkinson's.
Ohnuma, K., Kishita, Y., Nyuzuki, H., Kohda, M., Ohtsu, Y., Takeo, S., Asano, T., Sato-Miyata, Y., Ohtake, A., Murayama, K., Okazaki, Y. and Aigaki, T. (2020). Ski3/TTC37 deficiency associated with trichohepatoenteric syndrome causes mitochondrial dysfunction in Drosophila. FEBS Lett. PubMed ID: 32294252
Tetratricopeptide repeat protein 37 (TTC37) is a causative gene of trichohepatoenteric syndrome (THES). However, little is known about the pathogenesis of this disease. This study characterized the phenotype of a Drosophila model in which ski3, a homolog of TTC37, is disrupted. The mutant flies are pupal lethal, and the pupal lethality is partially rescued by transgenic expression of wild-type ski3 or human TTC37. The mutant larvae show growth retardation, heart arrhythmia, triacylglycerol accumulation, and aberrant metabolism of glycolysis and the TCA cycle. Moreover, mitochondrial membrane potential and respiratory chain complex activities are significantly reduced in the mutants. These results demonstrate that ski3 deficiency causes mitochondrial dysfunction, which may underlie the pathogenesis of THES.

Thursday, June 4th - Gonads

Kawaguchi, S., Ueki, M. and Kai, T. (2020). Drosophila MARF1 ensures proper oocyte maturation by regulating nanos expression. PLoS One 15(4): e0231114. PubMed ID: 32243476
Meiosis and oocyte maturation are tightly regulated processes. The meiosis arrest female 1 (MARF1) gene is essential for meiotic progression in animals; however, its detailed function remains unclear. This study examined the molecular mechanism of dMarf1, a Drosophila homolog of MARF1 encoding an OST and RNA Recognition Motif (RRM) -containing protein for meiotic progression and oocyte maturation. Although oogenesis progressed in females carrying a dMarf1 loss-of-function allele, the dMarf1 mutant oocytes were found to contain arrested meiotic spindles or disrupted microtubule structures, indicating that the transition from meiosis I to II was compromised in these oocytes. The expression of the full-length dMarf1 transgene, but none of the variants lacking the OST and RRM motifs or the 47 conserved C-terminal residues among insect groups, rescued the meiotic defect in dMarf1 mutant oocytes. These results indicate that these conserved residues are important for dMarf1 function. Immunoprecipitation of Myc-dMarf1 revealed that several mRNAs are bound to dMarf1. Of those, the protein expression of nanos (nos), but not its mRNA, was affected in the absence of dMarf1. In the control, the expression of Nos protein became downregulated during the late stages of oogenesis, while it remained high in dMarf1 mutant oocytes. It is proposed that dMarf1 translationally represses nos by binding to its mRNA. Furthermore, the downregulation of Nos induces cycB expression, which in turn activates the CycB/Cdk1 complex at the onset of oocyte maturation.
Meng, C., Tian, G., Xu, C., Li, X., Zhang, Y., Wang, Y., Qin, J., Fok, E. K. L., Hinton, B. T., Mak, K. K., Shum, W. W., Chan, W. Y. and Xia, Y. (2020). Hippo kinases MST1 and MST2 control the differentiation of the epididymal initial segment via the MEK-ERK pathway. Cell Death Differ. PubMed ID: 32332916
Although the roles of the Hippo pathway in organogenesis and tumorigenesis have been well studied in multiple organs, its role in sperm maturation and male fertility has not been investigated. The initial segment (IS) of the epididymis plays a critical role in sperm maturation. IS differentiation is governed by ERK1/2 (see Drosophila Rolled), but the mechanisms of ERK1/2 activation in IS are not fully understood. This study shows that double knockout (dKO) of mammalian sterile 20-like kinases 1 and 2 (Mst1 and Mst2), homologs of Hippo in Drosophila, in the epididymal epithelium led to male infertility in mice. Sperm in the cauda epididymides of mutant mice were immotile with flagellar angulation and severely disorganized structures. Loss of Mst1/2 activated YAP and increased proliferation and cell death in all the segments of epididymis. The mutant mice showed substantially suppressed MEK/ERK signaling in the IS and failed IS differentiation. Deletion of Yap (see Drosophila Yorkie) restored the reduced MEK/ERK signaling, and partially rescued the defective IS differentiation and fertility in Mst1/2 dKO mice. These results demonstrate that YAP inhibits the MEK/ERK pathway in IS epithelial cells, and MST1/2 control IS differentiation and fertility at least partially by repressing YAP. Taken together, the Hippo pathway is essential for sperm maturation and male fertility.
Duan, T., Kitzman, S. C. and Geyer, P. K. (2020). Survival of Drosophila germline stem cells requires the chromatin binding protein Barrier-to-autointegration factor. Development. PubMed ID: 32345742
The nuclear lamina (NL) is an extensive protein network that underlies the inner nuclear envelope. This network includes LAP2-emerin-MAN1-domain (LEM-D) proteins that associate with the chromatin and DNA binding protein Barrier-to-autointegration factor (BAF). this study investigated the partnership between three NL Drosophila LEM-D proteins and BAF. In most tissues, only D-emerin/Otefin is required for NL enrichment of BAF, revealing an unexpected dependence on a single LEM-D protein. Prompted by these observations, BAF contributions were studied in the ovary, a tissue where D-emerin/Otefin function is essential. Berm cell-specific BAF knockdown causes phenotypes that mirror d-emerin/otefin mutants. Loss of BAF disrupts NL structure, blocks differentiation and promotes germ cell loss, phenotypes that are partially rescued by inactivation of the ATR and Chk2 kinases. These data suggest that similar to d-emerin/otefin mutants, BAF depletion activates the NL checkpoint that causes germ cell loss. Taken together, these findings provide evidence for a prominent NL partnership between the LEM-D protein D-emerin/Otefin and BAF, revealing that BAF functions with this partner in the maintenance of an adult stem cell population.
Wang, F., Wang, K., Forknall, N., Patrick, C., Yang, T., Parekh, R., Bock, D. and Dickson, B. J. (2020). Neural circuitry linking mating and egg laying in Drosophila females. Nature 579(7797): 101-105. PubMed ID: 32103180
Mating and egg laying are tightly cooordinated events in the reproductive life of all oviparous females. Oviposition is typically rare in virgin females but is initiated after copulation. This study identified the neural circuitry that links egg laying to mating status in Drosophila melanogaster. Activation of female-specific oviposition descending neurons (oviDNs) is necessary and sufficient for egg laying, and is equally potent in virgin and mated females. After mating, sex peptide-a protein from the male seminal fluid-triggers many behavioural and physiological changes in the female, including the onset of egg laying. Sex peptide is detected by sensory neurons in the uterus, and silences these neurons and their postsynaptic ascending neurons in the abdominal ganglion. This study shows that these abdominal ganglion neurons directly activate the female-specific pC1 neurons. GABAergic (gamma-aminobutyric-acid-releasing) oviposition inhibitory neurons (oviINs) mediate feed-forward inhibition from pC1 neurons to both oviDNs and their major excitatory input, the oviposition excitatory neurons (oviENs). By attenuating the abdominal ganglion inputs to pC1 neurons and oviINs, sex peptide disinhibits oviDNs to enable egg laying after mating. This circuitry thus coordinates the two key events in female reproduction: mating and egg laying.
Knapp, E. M., Li, W., Singh, V. and Sun, J. (2020). Nuclear receptor Ftz-f1 promotes follicle maturation and ovulation partly via bHLH/PAS transcription factor Sim. Elife 9. PubMed ID: 32338596
The NR5A-family nuclear receptors are highly conserved and function within the somatic follicle cells of the ovary to regulate folliculogenesis and ovulation in mammals; however, their roles in Drosophila ovaries are largely unknown. This study discovered that Ftz-f1, one of the NR5A nuclear receptors in Drosophila, is transiently induced in follicle cells in late stages of oogenesis via ecdysteroid signaling. Genetic disruption of Ftz-f1 expression prevents follicle cell differentiation into the final maturation stage, which leads to anovulation. In addition, it was demonstrated that the bHLH/PAS transcription factor Single-minded (Sim) acts as a direct target of Ftz-f1 to promote follicle cell differentiation/maturation and that Ftz-f1's role in regulating Sim expression and follicle cell differentiation can be replaced by its mouse homolog steroidogenic factor 1 (mSF-1). This work provides new insight into the regulation of follicle maturation in Drosophila and the conserved role of NR5A nuclear receptors in regulating folliculogenesis and ovulation.
Hopkins, B. R., Sepil, I. and Wigby, S. (2020). Structural variation in Drosophila melanogaster spermathecal ducts and its association with sperm competition dynamics. R Soc Open Sci 7(3): 200130. PubMed ID: 32269825
The ability of female insects to retain and use sperm for days, months, or even years after mating requires specialized storage organs in the reproductive tract. In most orders, these organs include a pair of sclerotized capsules known as spermathecae. This study reports that some Drosophila melanogaster females exhibit previously uncharacterized structures within the distal portion of the muscular duct that links a spermatheca to the uterus.These 'spermathecal duct presences' (SDPs) may form in either or both ducts and can extend from the duct into the sperm-storing capsule itself. It was further found that the incidence of SDPs varies significantly between genotypes, but does not change significantly with the age or mating status of females, the latter indicating that SDPs are not composed of or stimulated by sperm or male seminal proteins. SDPs affect neither the number of first male sperm held in a spermatheca nor the number of offspring produced after a single mating. However, evidence was found that SDPs are associated with a lack of second male sperm in the spermathecae after females remate. This raises the possibility that SDPs provide a mechanism for variation in sperm competition outcome among females.

Wednesday, May 3rd - Adult neural development and function

Menegazzi, P., Beer, K., Grebler, V., Schlichting, M., Schubert, F. K. and Helfrich-Forster, C. (2020). A Functional Clock Within the Main Morning and Evening Neurons of D. melanogaster Is Not Sufficient for Wild-Type Locomotor Activity Under Changing Day Length. Front Physiol 11: 229. PubMed ID: 32273848
A major challenge for all organisms that live in temperate and subpolar regions is to adapt physiology and activity to different photoperiods. A long-standing model assumes that there are morning (M) and evening (E) oscillators with different photoreceptive properties that couple to dawn and dusk, respectively, and by this way adjust activity to the different photoperiods. In the fruit fly Drosophila melanogaster, M and E oscillators have been localized to specific circadian clock neurons in the brain. This study investigated under different photoperiods the activity pattern of flies expressing the clock protein Period (Per) only in subsets of M and E oscillators. All fly lines that expressed Per only in subsets of the clock neurons had difficulties to track the morning and evening in a wild-type manner. The lack of the E oscillators advanced M activity under short days, whereas the lack of the M oscillators delayed E activity under the same conditions. In addition, it was found that flies expressing Per only in subsets of clock neurons showed higher activity levels at certain times of day or night, suggesting that M and E clock neurons might inhibit activity at specific moments throughout the 24 h. Altogether, this study shows that the proper interaction between all clock cells is important for adapting the flies' activity to different photoperiods and these findings are discussed in the light of the current literature.
Lazar, A. A. and Yeh, C. H. (2020). A molecular odorant transduction model and the complexity of spatio-temporal encoding in the Drosophila antenna. PLoS Comput Biol 16(4): e1007751. PubMed ID: 32287275
Many odorant receptors remain only partially characterized, and the odorant transduction process and the axon hillock spiking mechanism of the olfactory sensory neurons (OSNs) have yet to be fully determined. This study has advanced a comprehensive model of fruit fly OSNs as a cascade consisting of an odorant transduction process (OTP) and a biophysical spike generator (BSG). Odorant identity and concentration were modeled using an odorant-receptor binding rate tensor, modulated by the odorant concentration profile, and an odorant-receptor dissociation rate tensor, and quantitatively describe the mechanics of the molecular ligand binding/dissociation of the OTP. The resulting model of the Drosophila antenna provides a foundation for understanding the neural code of both odorant identity and odorant concentration and advances the state-of-the-art in a number of ways. First, it quantifies on the molecular level the spatio-temporal level of complexity of the transformation taking place in the antennae. The concentration-dependent spatio-temporal code at the output of the antenna circuits determines the level of complexity of olfactory processing in the downstream neuropils, such as odorant recognition and olfactory associative learning. Second, the model is biologically validated using multiple electrophysiological recordings. Third, the model demonstrates that the currently available data for odorant-receptor responses only enable the estimation of the affinity of the odorant-receptor pairs. The odorant-dissociation rate is only available for a few odorant-receptor pairs. Finally, the model calls for new experiments for massively identifying the odorant-receptor dissociation rates of relevance to flies.
Kumar, A., Tauxe, G. M., Perry, S., Scott, C. A., Dahanukar, A. and Ray, A. (2020). Contributions of the Conserved Insect Carbon Dioxide Receptor Subunits to Odor Detection. Cell Rep 31(2): 107510. PubMed ID: 32294446
The CO2 receptor in mosquitoes is broadly tuned to detect many diverse odorants. The receptor consists of three subunits (Gr1, Gr2, and Gr3) in mosquitoes but only two subunits in Drosophila: Gr21a (Gr1 ortholog) and Gr63a (Gr3 ortholog). This study demonstrates that Gr21a is required for CO2 responses in Drosophila, as has been shown for Gr63a. Next, a Drosophila double mutant for Gr21a and Gr63a was generated, and in this background, combinations of Aedes Gr1, Gr2, and Gr3 genes were functionally expressed in the CO2 empty neuron. Only two subunits, Gr2 and Gr3, suffice for response to CO2. Addition of Gr1 increases sensitivity to CO2, whereas it decreases the response to pyridine. The inhibitory effect of the antagonist isobutyric acid is observed upon addition of Gr1. Gr1 therefore increases the diversity of ligands of the receptor and modulates the response of the receptor complex.
Lacin, H., Williamson, W. R., Card, G. M., Skeath, J. B. and Truman, J. W. (2020). Unc-4 acts to promote neuronal identity and development of the take-off circuit in the Drosophila CNS. Elife 9. PubMed ID: 32216875
The Drosophila ventral nerve cord (VNC) is composed of thousands of neurons born from a set of individually identifiable stem cells. The VNC harbors neuronal circuits required to execute key behaviors, such as flying and walking. Leveraging the lineage-based functional organization of the VNC, this study investigated the developmental and molecular basis of behavior by focusing on lineage-specific functions of the homeodomain transcription factor, Unc-4. Unc-4 was found to function in lineage 11A to promote cholinergic neurotransmitter identity and suppress the GABA fate. In lineage 7B, Unc-4 promotes proper neuronal projections to the leg neuropil and a specific flight-related take-off behavior. It was also uncovered that Unc-4 acts peripherally to promote proprioceptive sensory organ development and the execution of specific leg-related behaviors. Through time-dependent conditional knock-out of Unc-4, it was found that its function is required during development, but not in the adult, to regulate the above events.
Lee, Y. J., Yang, C. P., Miyares, R. L., Huang, Y. F., He, Y., Ren, Q., Chen, H. M., Kawase, T., Ito, M., Otsuna, H., Sugino, K., Aso, Y., Ito, K. and Lee, T. (2020). Conservation and divergence of related neuronal lineages in the Drosophila central brain. Elife 9. PubMed ID: 32255422
Wiring a complex brain requires many neurons with intricate cell specificity, generated by a limited number of neural stem cells. Drosophila central brain lineages are a predetermined series of neurons, born in a specific order. To understand how lineage identity translates to neuron morphology, this study mapped 18 Drosophila central brain lineages. While large aggregate differences between lineages were found, shared patterns of morphological diversification were discovered. Lineage identity plus Notch-mediated sister fate govern primary neuron trajectories, whereas temporal fate diversifies terminal elaborations. Further, morphological neuron types may arise repeatedly, interspersed with other types. Despite the complexity, related lineages produce similar neuron types in comparable temporal patterns. Different stem cells even yield two identical series of dopaminergic neuron types, but with unrelated sister neurons. Together, these phenomena suggest that straightforward rules drive incredible neuronal complexity, and that large changes in morphology can result from relatively simple fating mechanisms.
Lerner, H., Rozenfeld, E., Rozenman, B., Huetteroth, W. and Parnas, M. (2020). Differential Role for a Defined Lateral Horn Neuron Subset in Naive Odor Valence in Drosophila. Sci Rep 10(1): 6147. PubMed ID: 32273557
Value coding of external stimuli in general, and odor valence in particular, is crucial for survival. In flies, odor valence is thought to be coded by two types of neurons: mushroom body output neurons (MBONs) and lateral horn (LH) neurons. MBONs are classified as neurons that promote either attraction or aversion, but not both, and they are dynamically activated by upstream neurons. This dynamic activation updates the valence values. In contrast, LH neurons receive scaled, but non-dynamic, input from their upstream neurons. It remains unclear how such a non-dynamic system generates differential valence values. Recently, PD2a1/b1 LH neurons were demonstrated to promote approach behavior at low odor concentration in starved flies. This study demonstrates that at high odor concentrations, these same neurons contribute to avoidance in satiated flies. The contribution of PD2a1/b1 LH neurons to aversion is context dependent. It is diminished in starved flies, although PD2a1/b1 neural activity remains unchanged, and at lower odor concentration. In addition, PD2a1/b1 aversive effect develops over time. Thus, these results indicate that, even though PD2a1/b1 LH neurons transmit hard-wired output, their effect on valence can change. Taken together, it is suggested that the valence model described for MBONs does not hold for LH neurons.

Tuesday, June 2nd - Signaling

Cruz, J., Martin, D. and Franch-Marro, X. (2020). Egfr Signaling Is a Major Regulator of Ecdysone Biosynthesis in the Drosophila Prothoracic Gland. Curr Biol 30(8): 1547-1554. PubMed ID: 32220314
Understanding the mechanisms that determine final body size of animals is a central question in biology. In animals with determinate growth, such as mammals or insects, the size at which the immature organism transforms into the adult defines the final body size, as adult individuals do not grow. In Drosophila, the growth period ends when the immature larva undergoes the metamorphic transition to develop the mature adult. This metamorphic transition is triggered by a sharp increase of the steroid ecdysone, synthetized in the prothoracic gland (PG), that occurs at the end of the third instar larvae (L3). It is widely accepted that ecdysone biosynthesis in Drosophila is mainly induced by the activation of tyrosine kinase (RTK) Torso by the prothoracicotropic hormone (Ptth) produced into two pairs of neurosecretory cells that project their axons onto the PG. However, the fact that neither Ptth nor torso-null mutant animals arrest larval development but only present a delay in the larva-pupa transition mandates for a reconsideration of the conventional model. This study shows that Egfr signaling, rather than Ptth/torso, is the major contributor of ecdysone biosynthesis in Drosophila. Egfr signaling was found to be activated in the PG in an autocrine mode by the EGF ligands spitz and vein, which in turn are regulated by the levels of ecdysone. This regulatory positive feedback loop ensures the production of ecdysone to trigger metamorphosis by a progressive Egfr-dependent activation of MAPK/ERK pathway, thus determining the animal final body size.
Li, Y., Romey-Glusing, R., Tahan Zadeh, N., von Frieling, J., Hoffmann, J., Huebbe, P., Bruchhaus, I., Rimbach, G., Fink, C. and Roeder, T. (2020). Furbellow (Brown Algae) Extract Increases Lifespan in Drosophila by Interfering with TOR-Signaling. Nutrients 12(4). PubMed ID: 32331413
Algal products are well known for their health promoting effects. Nonetheless, an in depth understanding of the underlying molecular mechanisms is still only fragmentary. This study shows that aqueous furbelow extracts (brown algae, Saccorhiza polyschides) lengthen the life of both sexes of the fruit fly Drosophila melanogaster substantially, if used as nutritional additives to conventional food. This life prolonging effect became even more pronounced in the presence of stressors, such as high-fat dieting of living under drought conditions. Application of the extracts did not change food intake, excretion, or other major physiological parameters. Nevertheless, effects on the intestinal microbiota were observed, leading to an increased species richness, which is usually associated with healthy conditions. Lifespan extension was not observed in target of rapamycin (TOR)-deficient animals, implying that functional TOR signaling is necessary to unfold the positive effects of brown algae extract (BAE) on this important trait. The lack of life lengthening in animals with deregulated TOR signaling exclusively targeted to body fat showed that this major energy storage organ is instrumental for transmitting these effects. In addition, expression of Imaginal morphogenesis protein-Late 2 (Imp-L2), an effective inhibitor of insulin signaling implies that BAE exerts their positive effects through interaction with the tightly interwoven TOR- and insulin-signaling systems, although insulin levels were not directly affected by this intervention.
D'Ignazio, L., Shakir, D., Batie, M., Muller, H. A. and Rocha, S. (2020). HIF-1beta Positively Regulates NF-kappaB Activity via Direct Control of TRAF6. Int J Mol Sci 21(8). PubMed ID: 32344511
NF-kappaB signalling is crucial for cellular responses to inflammation but is also associated with the hypoxia response. NF-kappaB and hypoxia inducible factor (HIF) transcription factors possess an intense molecular crosstalk. Although it is known that HIF-1alpha modulates NF-kappaB transcriptional response, very little is understood regarding how HIF-1beta contributes to NF-kappaB signalling. This study demonstrates that HIF-1beta is required for full NF-kappaB activation in cells following canonical and non-canonical stimuli. HIF-1beta was found to specifically control TRAF6 expression in human cells but also in Drosophila melanogaster. HIF-1beta binds to the TRAF6 gene and controls its expression independently of HIF-1alpha. Furthermore, exogenous TRAF6 expression is able to rescue all of the cellular phenotypes observed in the absence of HIF-1beta. These results indicate that HIF-1beta is an important regulator of NF-kappaB with consequences for homeostasis and human disease.
Gonzalez-Mendez, L., Gradilla, A. C., Sanchez-Hernandez, D., Gonzalez, E., Aguirre-Tamaral, A., Jimenez-Jimenez, C., Guerra, M., Aguilar, G., Andres, G., Falcon-Perez, J. M. and Guerrero, I. (2020). Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc. EMBO J: e103629. PubMed ID: 32311148
Hedgehog (Hh) signal molecules play a fundamental role in development, adult stem cell maintenance and cancer. Hh can signal at a distance, and it has been proposed that its graded distribution across Drosophila epithelia is mediated by filopodia-like structures called cytonemes. Hh reception by Patched (Ptc) happens at discrete sites along presenting and receiving cytonemes, reminiscent of synaptic processes. This study show that a vesicle fusion mechanism mediated by SNARE proteins is required for Ptc placement at contact sites. Transport of Ptc to these sites requires multivesicular bodies (MVBs) formation via ESCRT machinery, in a manner different to that regulating Ptc/Hh lysosomal degradation after reception. These MVBs include extracellular vesicle (EV) markers and, accordingly, Ptc is detected in the purified exosomal fraction from cultured cells. Blockage of Ptc trafficking and fusion to basolateral membranes result in low levels of Ptc presentation for reception, causing an extended and flattened Hh gradient.
Li, S., Tian, A., Li, S., Han, Y., Wang, B. and Jiang, J. (2020). Gilgamesh (Gish)/CK1gamma regulates tissue homeostasis and aging in adult Drosophila midgut. J Cell Biol 219(4). PubMed ID: 32328627
Adult tissues and organs rely on resident stem cells to generate new cells that replenish damaged cells. To maintain homeostasis, stem cell activity needs to be tightly controlled throughout the adult life. This study shows that the membrane-associated kinase Gilgamesh (Gish)/CK1gamma maintains Drosophila adult midgut homeostasis by restricting JNK pathway activity and that Gish is essential for intestinal stem cell (ISC) maintenance under stress conditions. Inactivation of Gish resulted in aberrant JNK pathway activation and excessive production of multiple cytokines and growth factors that drive ISC overproliferation. Mechanistically, Gish restricts JNK activation by phosphorylating and destabilizing a small GTPase, Rho1. Interestingly, this study found that Gish expression is down-regulated in aging guts and that increasing Gish activity in aging guts can restore tissue homeostasis. Hence, this study identifies Gish/CK1gamma as a novel regulator of Rho1 and gatekeeper of tissue homeostasis whose activity is compromised in aging guts.
Humphries, A. C., Narang, S. and Mlodzik, M. (2020). Mutations associated with human neural tube defects display disrupted planar cell polarity in Drosophila. Elife 9. PubMed ID: 32234212
Planar cell polarity (PCP) and neural tube defects (NTDs) are linked, with a subset of NTD patients found to harbor mutations in PCP genes, but there is limited data on whether these mutations disrupt PCP signaling in vivo. The core PCP gene Van Gogh (Vang), Vangl1/2 in mammals, is the most specific for PCP. This study addressed potential causality of NTD-associated Vangl1/2 mutations, from either mouse or human patients, in Drosophila allowing intricate analysis of the PCP pathway. Introducing the respective mammalian mutations into Drosophila Vang revealed defective phenotypic and functional behaviors, with changes to Vang localization, post-translational modification, and mechanistic function, such as its ability to interact with PCP effectors. These findings provide mechanistic insight into how different mammalian mutations contribute to developmental disorders and strengthen the link between PCP and NTD. Importantly, analyses of the human mutations revealed that each is a causative factor for the associated NTD.

Monday, June 1st - Adult Physiology

Alvarez-Rendon, J. P. and Riesgo-Escovar, J. R. (2020). Circadian and Rhythmic-Related Behavioral Co-Morbidities of the Diabetic State in Drosophila melanogaster. Gen Comp Endocrinol: 113477. PubMed ID: 32240709
Circadian phenomena rule many activities of life on earth. Disruptions in circadian rhythmicity and rhythms have been recognized as a contributing factor for diseased states, for instance metabolic disruptions like diabetes. Diabetes develops as a consequence of faulty insulin pathway signaling, either by lack of insulin production (diabetes type I), or by loss of responsiveness in target tissues (diabetes type 2). This work used the model organism Drosophila melanogaster with three different mutant hypomorphic conditions at different levels of the insulin pathway. The insulin pathway is a very evolutionarily conserved pathway. These different diabetic conditions were studied as a source of circadian rhythm abnormalities and circadian-related co-morbidities. This is done by studying circadian rhythmicity, activity, sleep and sleep structure, and feeding behavior. Results show that flies with impaired insulin signaling show circadian rhythm and rhythmic-related co-morbidities, especially female flies, as a consequence of the diabetic state. The most extreme disruptions occur in flies with impaired insulin receptor signaling, which stands at the beginning of the insulin pathway, in principle affecting most if not all aspects of this pathway. This work shows that defective insulin signaling is a source of circadian rhythm and rhythmic related co-morbidities.
Bawa, S., Brooks, D. S., Neville, K. E., Tipping, M., Sagar, M. A., Kollhoff, J. A., Chawla, G., Geisbrecht, B. V., Tennessen, J. M., Eliceiri, K. W. and Geisbrecht, E. R. (2020). Drosophila TRIM32 cooperates with glycolytic enzymes to promote cell growth. Elife 9. PubMed ID: 32223900
Cell growth and/or proliferation may require the reprogramming of metabolic pathways, whereby a switch from oxidative to glycolytic metabolism diverts glycolytic intermediates towards anabolic pathways. This study identified a novel role for TRIM32 in the maintenance of glycolytic flux mediated by biochemical interactions with the glycolytic enzymes Aldolase and Phosphoglycerate mutase. Loss of Drosophila TRIM32, encoded by thin (tn), shows reduced levels of glycolytic intermediates and amino acids. This altered metabolic profile correlates with a reduction in the size of glycolytic larval muscle and brain tissue. Consistent with a role for metabolic intermediates in glycolysis-driven biomass production, dietary amino acid supplementation in tn mutants improves muscle mass. Remarkably, TRIM32 is also required for ectopic growth - loss of TRIM32 in a wing disc-associated tumor model reduces glycolytic metabolism and restricts growth. Overall, these results reveal a novel role for TRIM32 for controlling glycolysis in the context of both normal development and tumor growth.
Huang, R., Song, T., Su, H., Lai, Z., Qin, W., Tian, Y., Dong, X. and Wang, L. (2020). High-fat diet enhances starvation-induced hyperactivity via sensitizing hunger-sensing neurons in Drosophila. Elife 9. PubMed ID: 32324135
The function of the central nervous system to regulate food intake can be disrupted by sustained metabolic challenges such as high-fat diet (HFD), which may contribute to various metabolic disorders. Previous work has shown that a group of octopaminergic (OA) neurons mediated starvation-induced hyperactivity, an important aspect of food-seeking behavior. This study found that HFD specifically enhances this behavior. Mechanistically, HFD increases the excitability of these OA neurons to a hunger hormone named adipokinetic hormone (AKH), via increasing the accumulation of AKH receptor (AKHR) in these neurons. Upon HFD, excess dietary lipids are transported by a lipoprotein LTP to enter these OA(+)AKHR(+) neurons via the cognate receptor LpR1, which in turn suppresses autophagy-dependent degradation of AKHR. Taken together, this study has uncovered a mechanism that links HFD, neuronal autophagy, and starvation-induced hyperactivity, providing insight in the reshaping of neural circuitry under metabolic challenges and the progression of metabolic diseases.
Li, S., Tian, A., Li, S., Han, Y., Wang, B. and Jiang, J. (2020). Gilgamesh (Gish)/CK1gamma regulates tissue homeostasis and aging in adult Drosophila midgut. J Cell Biol 219(4). PubMed ID: 32328627
Adult tissues and organs rely on resident stem cells to generate new cells that replenish damaged cells. To maintain homeostasis, stem cell activity needs to be tightly controlled throughout the adult life. This study shows that the membrane-associated kinase Gilgamesh (Gish)/CK1gamma maintains Drosophila adult midgut homeostasis by restricting JNK pathway activity and that Gish is essential for intestinal stem cell (ISC) maintenance under stress conditions. Inactivation of Gish resulted in aberrant JNK pathway activation and excessive production of multiple cytokines and growth factors that drive ISC overproliferation. Mechanistically, Gish restricts JNK activation by phosphorylating and destabilizing a small GTPase, Rho1. Interestingly, Gish expression was found to be down-regulated in aging guts and that increasing Gish activity in aging guts can restore tissue homeostasis. Hence, this study identifies Gish/CK1gamma as a novel regulator of Rho1 and gatekeeper of tissue homeostasis whose activity is compromised in aging guts.
Bitner, K., Shahrestani, P., Pardue, E. and Mueller, L. D. (2020). Predicting death by the loss of intestinal function. PLoS One 15(4): e0230970. PubMed ID: 32287318
The ability to predict when an individual will die can be extremely useful for many research problems in aging. A technique for predicting death in the model organism, Drosophila melanogaster, has been proposed which relies on an increase in the permeability of the fly intestinal system, allowing dyes from the diet to permeate the body of the fly shortly before death. This study sought to verify this claim in a large cohort study using different populations of D. melanogaster and different dyes. Only about 50% of the individuals showed a visible distribution of dye before death. This number did not vary substantially with the dye used. Most flies that did turn a blue color before death did so within 24 hours of death. There was also a measurable effect of the dye on the fly mean longevity. These results would tend to limit the utility of this method depending on the application the method was intended for.
Hernandez-Gallardo, A. K. and Missirlis, F. (2020). Cellular iron sensing and regulation: Nuclear IRP1 extends a classic paradigm. Biochim Biophys Acta Mol Cell Res: 118705. PubMed ID: 32199885
The classic view is that iron regulatory proteins operate at the post-transcriptional level. Iron Regulatory Protein 1 (IRP1) shifts between an apo-form that binds mRNAs and a holo-form that harbors a [4Fe4S] cluster. The latter form is not considered relevant to iron regulation, but rather thought to act as a non-essential cytosolic aconitase. Recent work in Drosophila, however, shows that holo-IRP1 can also translocate to the nucleus, where it appears to downregulate iron metabolism genes, preparing the cell for a decline in iron uptake. The shifting of IRP1 between states requires a functional mitoNEET pathway that includes a glycogen branching enzyme for the repair or disassembly of IRP1's oxidatively damaged [3Fe4S] cluster. The new findings add to the notion that glucose metabolism is modulated by iron metabolism. Furthermore, it is proposed that ferritin ferroxidase activity participates in the repair of the IRP1 [3Fe4S] cluster leading to the hypothesis that cytosolic ferritin directly contributes to cellular iron sensing.
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